Syntaxin 6-mediated exosome secretion regulates enzalutamide resistance in prostate cancer

Abstract

Prostate cancer (PCa) deaths are typically the result of metastatic castration-resistant PCa (mCRPC). Recently, enzalutamide (Enz), an oral AR inhibitor, was approved for treating patients with mCRPC. Invariably, all PCa patients eventually develop resistance against Enz. Therefore, novel strategies aimed at overcoming Enz resistance are needed to improve the survival of PCa patients. The role of exosomes in drug resistance has not been fully elucidated in PCa. Therefore, we set out to better understand exosome’s role in the mechanism underlying Enz-resistant PCa. Results showed that Enz-resistant PCa cells (C4–2B, CWR-R1, and LNCaP) secreted significantly higher amounts of exosomes (2–4 folds) compared to Enz-sensitive counterparts. Inhibition of exosome biogenesis in resistant cells by GW4869 and dimethyl amiloride (DMA) strongly decreased their cell viability. Mechanistic studies revealed increased upregulation of syntaxin 6 as well as its increased colocalization with CD63 in Enz-resistant PCa cells compared to Enz-sensitive cells. Syntaxin 6 knockdown by specific siRNAs in Enz-resistant PCa cells (C4–2B and CWR-R1) resulted in reduced cell number and increased cell death in the presence of Enz. Furthermore, syntaxin 6 knockdown significantly reduced the exosome secretion in both Enz-resistant C4–2B and CWR-R1 cells. TCGA analysis showed increased syntaxin 6 expressions associated with higher Gleason score and decreased progression free survival in PCa patients. Importantly, IHC analysis showed significantly higher syntaxin 6 expression in cancer tissues from Enz treated patients compared to Enz naïve patients. Overall, syntaxin 6 plays an important role in the secretion of exosomes and increased survival of Enz-resistant PCa cells.

Introduction

Prostate cancer (PCa) is the most common non-cutaneous malignancy diagnosed in men in the United States with an estimated 174,650 new cases in 2019 and 31,620 deaths.¹ Androgen and androgen receptor (AR)-mediated signaling play a critical role in the development and progression of prostate cancer (PCa).^2,3 Therefore, ablation of androgens has been the frontline therapy for treating patients with locally advanced PCa, but in most cases, disease eventually progresses to an untreatable hormone-refractory stage. The treatment options for these men include chemotherapy, immunotherapy, and novel AR-directed agents, one of which is enzalutamide (Enz).

Enzalutamide (Xtandi®), an oral AR inhibitor, is approved for the treatment of metastatic castration-resistant PCa (mCRPC). It is unique as a second-generation AR antagonist in that it works via three different mechanisms. It does not exhibit any measurable agonistic activity, and in addition to acting as an AR blocker, it disrupts the translocation of the AR from the cytoplasm into the nucleus and impairs binding of the AR to the transcriptional complex. Despite its enhanced activity against the AR, however, approximately 20 to 40% of patients have no prostate-specific antigen (PSA) response.^4–6 Although median survival time for hormone-refractory PCa patients has significantly improved with Enz treatment, invariably all PCa patients eventually develop resistance against Enz and die from hormone-refractory PCa. Enz-resistant PCa cells also develop cross-resistance to abiraterone, an androgen synthesis inhibitor approved to treat metastatic hormone-refractory PCa.⁷ At present, Enz-resistant hormone-refractory PCa is a major clinical challenge, and novel strategies aimed at overcoming Enz resistance are urgently needed to improve the survival of PCa patients.

There have been a number of studies examining proposed mechanisms behind Enz-resistance. These mechanisms include upregulation of the AR and cytochrome P450 17α-hydroxylase/17,20-lyase, induction of AR splice variants, AR point mutations, upregulation of the glucocorticoid receptor by BET bromodomain and cortisol, activation of alternative oncogenic signaling pathways, neuroendocrine transformation, increased cellular plasticity through mutations in tumor suppressor genes, and immune evasion via programmed death-ligand 1 upregulation.^8–11 However to date, there has been no study to evaluate the role that exosomes play in the development of Enz resistance in PCa cells.

Exosomes are extracellular vesicles ~30–150 nm in size that are secreted by all cell types. Their biogenesis involves formation and fusion of clathrin-coated vesicles (CCV) with the endosome; inward budding of the membrane of early endosomes, cargo loading leading to formation of multi-vesicular endosomes (MVEs) with exosomes inside; and MVE fusion with the plasma membrane and exosome release. Exosomes are present in all biofluids including blood, urine, saliva, milk, cerebrospinal fluid and semen.¹² Exosomes are loaded with unique cargo, including RNA, protein, and metabolites that could predict the cell of their origin and the cellular physiologic and metabolic state.^13–16 Exosomes released by cells under stressful conditions or pathologic states are different from those released under normal physiologic and metabolic conditions.^{13,15, 17–19} For example, exosomes secreted by cells in response to oxidative stress were loaded with unique proteins representative of oxidative physiological state.^15,17,18 Thus, for this reason, exosomes are being extensively studied for diagnosis and prognosis of several diseases, including cancer, diabetes, and atherosclerosis.^16,19,20 Specifically, exosomes and their role in drug-resistant malignancy have been explored in breast cancer, pancreatic cancer, glioblastoma, hepatocellular carcinoma, and colorectal cancer.²¹ Not only have they been shown to transmit resistance through transference of miRNA, but other studies have demonstrated that exosomes are loaded with drug for export out of the cell.²² However, no study has yet examined whether exosomes could mediate Enz resistance in PCa cells.

Central to exosomal trafficking are soluble N-ethylmaleimide attachment protein receptor (SNARE) proteins. These proteins are tail-anchored membrane proteins involved in membrane fusion events along the secretory pathway. There are two types of SNAREs, classified as t-SNAREs and v-SNAREs, with t-SNAREs being found on the target membrane and v-SNAREs on the vesicle membrane. Syntaxin 6 is among those t-SNAREs particularly important in vesicle fusion. Syntaxin 6 is primarily located on the trans-Golgi network, with the remaining protein located in endosomes and transport vesicles.²³ Under conditions of overexpression, it can be found diffusely spread within the cytoplasm.²⁴ Gene expression analyses have demonstrated that syntaxin 6 is upregulated in numerous cancers including breast, colon, liver, pancreatic, prostate, bladder, skin, testicular, tongue, cervical, lung and gastric cancers.²⁵ In cancerous cells, syntaxin 6 has previously been shown to regulate chemotactic cell migration through integrin trafficking to the cell surface, promoting activation of focal adhesion kinase (FAK).²⁵ Syntaxin 6 has also been identified as a common p53 target and as a promoter of cancer cell growth in a p53-dependent manner.²⁶ Investigators found that p53 mutants in human esophageal cancer could induce syntaxin 6, which would then counter the anoikis apoptotic pathway.²⁷ In our previous work, we demonstrated that syntaxin 6 is an important biomarker in predicting overall survival in patients with papillary renal cell carcinoma.²⁸ In the present study, we set out to determine its role in the development of Enz-resistant PCa through the utilization of exosomal pathways.

Materials and Methods

Cells and Reagents

C4–2B Enz-sensitive cells (C4–2B-S) and C4–2B Enz-resistant cells (C4–2B-R) were obtained from Dr. Devasis Chatterjee lab at Brown University, Rhode Island. Enz-sensitive and resistant CWR-R1 cells (CWR-R1-S and CWR-R1-R, respectively), along with Enz-sensitive and resistant LNCaP cells (LNCaP-S and LNCaP-R, respectively) were obtained from the Vander Griend lab at the University of Chicago, Illinois.²⁹ All cell lines were cultured at 37°C in a 5% CO2 humidified environment as adherent monolayer in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin G and 100 μg/ml streptomycin sulfate. The resistant cell lines were cultured in 20 uM enzalutamide, dissolved in dimethyl sulfoxide (DMSO). Enzalutamide was purchased from Selleck Chemicals MDV3100 (Cat #S1250). The antibody for syntaxin 6 (Cat#2869; RRID:AB_2196500), as well as the Alexa Fluor antibodies (Cat#4413S: Anti-rabbit IgG Alexa Fluor555 and Cat#4410S Anti-mouse IgG Alexa Fluor 647) used in immunofluorescence (IF), were from Cell Signaling (Beverly, MA). The antibodies for CD63 (Cat#59479; RRID: AB_940915) and alpha-tubulin (Cat#7291; RRID: AB_2241126) were from Abcam (Cambridge, MA). The syntaxin 6 antibody (Cat#56656, RRID: AB_945728) used for immunohistochemistry (IHC) was also from Abcam. Enhanced chemiluminescence (ECL) detection system was from Bio-Rad (Hercules, CA). All other reagents were obtained in their commercially available highest purity grade.

Exosome Isolation

Cells were cultured for 24 hrs; thereafter, fresh media supplemented with 10% exosome-depleted FBS was added and cells were cultured for 48 hrs. Subsequently, conditioned media was harvested and exosomes were isolated by serial centrifugation. In brief, the collected cell culture media was centrifuged at 500 g at 4°C for 5 minutes to remove detached cells. The media was then centrifuged at 2,000 g at 4°C for 5 minutes to remove cellular debris. Then the supernatant was collected and passed through 0.22 μm filters. The filtrate was then concentrated using Pierce concentrators (150K MWCO/20 ml) by centrifuging at 3,000 g for 15 min. The supernatants were then subjected to ultracentrifugation at 100,000 g for 70 minutes (L-80 Ultracentrifuge, 70.1 Ti fixed angel rotor, Beckman Coulter). Finally, the pelleted exosomes were re-suspended in DPBS and stored at 4°C until further use.

Western Blotting

Cells were lysed with RIPA lysis buffer and Halt Protease/Phosphatase inhibitor (ThermoFisher Scientific, Rockford, IL). Thereafter, 50–70 μg of protein lysate per sample was denatured in 2x sample buffer and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 12% Tris–glycine gel (as required based upon the protein molecular weight). The separated proteins were transferred on to nitrocellulose membrane followed by blocking with 5% non-fat milk powder (w/v) in Tris-buffered saline (10 mM Tris–HCl, pH 7.5, 100 mM NaCl, 0.1% Tween 20) for 1 hr at room temperature. Membranes were probed for the protein levels of desired molecules using specific primary antibodies followed by the appropriate peroxidase-conjugated secondary antibody and visualized by ECL detection system. To ensure equal protein loading, each membrane was stripped and re-probed with appropriate loading control. The autoradiograms/bands were scanned using Adobe Photoshop 6.0 (Adobe Systems, San Jose, CA). Densitometry values were measured using ImageJ and presented below the bands as ‘fold change’ compared to control after loading control normalization.

Nanoparticle tracking analysis (NTA)

Quantification of the hydrodynamic diameter distribution and concentration of exosomes was performed using the Nanosight NS500 (Malvern Instruments, UK) equipped with a violet laser (405 nm) and running software version NTA3.2. The instrument was primed using phosphate buffered saline, pH 7.4 (PBS) and the temperature was maintained at 25°C. Accurate nanoparticle tracking was verified using 50 nm and 200 nm polystyrene nanoparticle standards (Malvern Instruments) prior to examination of the samples. Purified exosome samples were diluted in an appropriate volume of PBS such that there were 20–60 particles per field of view. Five measurements (60 seconds each) were obtained for each sample. Data are represented as the average result of these five measurements.

1D SDS-PAGE and mass spectrometry analysis

Exosomes were lysed with RIPA lysis buffer and Halt Protease/Phosphatase inhibitor (ThermoFisher Scientific, Rockford, IL). Next, exosomal lysate (50 μg) was electrophoresed on a 10% 1D SDS-PAGE and the gel was stained with Coomassie brilliant blue and cut into 5 slices. Gel slices were digested using a protocol modified from Lee et al.³⁰ Proteins were processed by LC/MS/MS at Proteomics and Metabolomics Shared Resource at Wake Forest Baptist Medical Center. The annotated human protein database from UniProtKB (20,161 entries, date; 10–1-2014) was used within Proteome Discoverer analytical software (v1.4, Thermo Scientific, Rockford, IL, USA) applying the generic MASCOT search algorithm. The protein subcellular localizations and functions were determined from the Ingenuity Systems software (Redwood City, CA, USA; http://www.ingenuity.com/index.html). Pathway analysis and network constructions were assembled using the Ingenuity software.

Confocal Microscopy

Cells were seeded onto 8-well chambered coverslips and incubated for 24 hrs in normal growth media. The media was removed and cells were fixed with formaldehyde/cold methanol and incubated overnight in primary antibody (anti-Syntaxin 6 or CD63). The cells were then washed and treated with secondary Alexa-Fluor 555 or 647 conjugated antibody and DAPI nuclear stain. Images were captured by Olympus FV1200 Laser Scanning Confocal Microscope. Images were quantified using ImageJ software.

Syntaxin 6 Knockdown

C4–2B-R and CWR-R1-R cells were seeded in 6 well plates one day prior to the transfection such that confluency at transfection was ~70%. The media was removed, and 2 ml fresh media containing syntaxin 6 siRNA (ON-TARGET plus Human syntaxin 6 siRNA-SMART pool, Catalog # L-017164–00-0010, Dharmacon) and transfection reagents (Catalog # T-2001–01 Horizon Discovery) were added drop-wise over the cells. Media was changed the next day. Cells were collected after 48 hrs and knockdown was confirmed by Western blotting.

Trypan assay

Cell number and cell death were counted using Trypan blue dye exclusion method. At the end of each mentioned time point, total cells (attached and floaters) were collected by a brief trypsinization and counted using hemocytometer. Trypan blue dye (0.4% in PBS) was used for assessing dead cells.

MTT assay

Cells were plated at a density of 2,000 cells/well in 96-well plate under standard culture conditions. The following day, the media was changed to include the indicated treatment. After 72 hrs, 20 μl of MTT (5 mg/ml stock) was added to media, and incubated for another 4 hrs at 37°C in a CO₂ incubator. At the end, media was removed and 100 μl of DMSO was added to each well. Color intensity was measured with absorbance at 560 nm. Background absorbance was measured at 650 nm. We duplicated the assay for all cell lines.

TCGA

RNA sequencing (RNA-Seq) data from 333 individuals within the Prostate Cancer cohort were obtained from the TCGA data portal (https://gdc.cancer.gov/). Clinical and pathologic data on TCGA patients were also downloaded from the Broad Institute TCGA GDAC Firehose.

Immunohistochemistry (IHC)

IHC was performed on formalin-fixed paraffin-embedded tissue sections from Enz-treated and Enz-naive patients obtained from the Prostate Cancer Biorepository Network (PCBN). Antigen unmasking was performed by heat-induced epitope retrieval using citrate buffer (10 mMol/L) at pH 6.0, and endogenous alkaline peroxidase (AP) activity was quenched by incubating with BLOXALL blocking solution for 10 minutes. Protein block was done by normal horse serum (2.5%) for 20 minutes. Then the sections were incubated with anti-syntaxin 6 antibody (1:400; mouse monoclonal) overnight at 4°C, followed by incubation with ImmPRESS AP reagent-anti-mouse secondary antibody for 30 minutes at room temperature. ImmPACTTM Vector® Red substrate (Vector Laboratories, Burlingame, CA) was used for stain development, and counterstain was done with hematoxylin. Microscopic examination was done to confirm the diagnosis and tumor grade. All the immunostained slides were scanned by NanoZoomer (Hamamatsu, Japan), and using the apps developed in VisioPharm, Syntaxin6 staining intensities were scored ranging from 0 to 4 with 0 representing the negative staining and 4 the strongest staining. In VisioPharm, the total area of ROI (region of interest) was measured for each specimen; and the areas of staining score 1 to 4 were also measured respectively, and then the ratios of the area for each score versus the total area were calculated.

Statistics

Statistical analysis was performed using GraphPad prism 7.0 (La Jolla, CA) or SigmaStat 4.0 and presented as mean±SEM. t-test was performed and a p value of ≤ 0.05 was accepted as statistically significant.

Results

Enz-resistant cells secrete more exosomes than Enz-sensitive cells

Exosomes from C4–2B-S, C4–2B-R, CWR-R1-S, CWR-R1-R, LNCaP-S, and LNCaP-R were analyzed for concentration and size distribution by nanoparticle tracking analysis (NTA). Data revealed that total exosome concentration per ml, after normalization for cell number, was significantly greater in all resistant cell lines as compared to the corresponding sensitive cell line (Figure 1). C4–2B-R secreted almost three times as many exosomes as C4–2B-S (P≤0.0001) (Figure 1A). CWR-R1-R secreted two times as many exosomes as CWR-R1-S (P ≤0.0001) (Figure 1B). LNCaP-R secreted over 3.5 times as many exosomes as LNCaP-S (P ≤0.0001) (Figure 1C). The average size of exosomes ranged from 135 to 145 nm. There was no significant difference in particle size between the sensitive and corresponding resistant cell lines.

Figure 1. Characterization of exosomes secreted by PCa cells.

(A-C) Exosomes secreted by Enz-resistant and -sensitive cells were isolated and analyzed for particle size distribution and concentration by NTA as described in methods, with mean +/− SEM Representative particle size distribution for exosomes is presented. Data represent mean ± SEM of five videos. (^*P ≤ 0.0001)

Exosomes from C4–2B-S and C4–2B-R cells were also analyzed for protein loading (Supplementary Figure 1). Exosomes secreted by C4–2B-R cells were loaded with a lesser number of proteins compared to exosomes secreted by C4–2B-S cells (Supplementary Figure 1A). We identified 381 proteins in Exo^{Enz-resistant} and 549 proteins in Exo^{Enz-sensitive}, which were tracked using Ingenuity Pathway Analysis. The proteins were categorized based on their sub-cellular or extra-cellular localization. As shown, in Supplementary Figure 1A, Exo^{Enz-sensitive} and in Exo^{Enz-resistant} were loaded with proteins primarily from the plasma membrane, cytoplasm, and extracellular space, with low percentages of proteins from the nucleus. Using IPA software, the identified proteins were grouped into networks of associated functions and canonical pathways. In Supplementary Figure 1B, we have presented the differences in the top 15 canonical pathways of Exo^{Enz-sensitive} and Exo^{Enz-resistant} associated proteins. The scores (−log [p value]) reflect the probabilities of such association occurring by chance, with the threshold value for significance set at 1.25. Supplementary Figures 1C and 1D how the “interactome” of the highest-scoring local connecting network and functional associations within these networks for Exo^{Enz-sensitive} and Exo^{Enz-resistant}, respectively. Direct interactions between identified proteins are indicated by solid lines and indirect connections are shown with broken lines.

Inhibition of exosome secretion and biogenesis lead to decreased cell survival in Enz-resistant PCa cells

With a significant increase in exosome secretion in the resistant cell lines, we then wanted to determine whether exosome secretion was critical for their survival. Therefore, we used two compounds that are known to affect exosome biogenesis and secretion, GW-4869 and 5-(N,N-Dimethyl)amiloride hydrochloride (DMA). GW4869, a neutral sphingomyelinase inhibitor, is the most widely used pharmacological agent for blocking exosome generation. Specifically, it inhibits the ceramide-mediated inward budding of multivesicular bodies (MVBs) and release of mature exosomes from MVBs.³¹ DMA is an inhibitor of H⁺/Na⁺ and Na⁺/Ca²⁺ exchangers, thus preventing the establishment of a calcium gradient that is essential for exosome release.³² We treated C4–2B-R, CWR-R1-R, and LNCaP-R cells with GW4869 (20uM) and DMA (1ug/mL). GW4869 led to a significant decrease in cell viability across all three resistant cell lines. In C4–2B-R, GW4869 led to a 38.6% (P ≤0.0001) decrease in viability compared to control (Figure 2A). In CWR-R1-R, GW4869 led to a 39.3% (P ≤0.0001) decrease in viability compared to control (Figure 2B). In LNCaP-R, GW4869 led to a 37.8% (p≤0.0001) decrease in viability compared to control (Figure 2C). DMA led to a significant decrease in cell viability in CWR-R1-R cells (P ≤0.0001) and LNCaP-R cells (P =0.004), but not C4–2B-R cells (P=0.2215) (Figure 2A-C).

Figure 2. Exosome biogenesis inhibitors decrease viability of Enz-resistant PCa cells.

(A-C) Enz-resistant (R) PCa cells were seeded at a density of 2 ×10³ cells/well in 96-well plates. After 24 hours of seeding, cells were treated with DMSO (vehicle), GW4869 (20 uM), or DMA, 5-(N,N-Dimethyl)amiloride hydrochloride(1 ug/mL). After 72 hrs, cell viability was assessed by MTT assay, with Mean +/− SEM. For each assay, we performed 5–10 technical replications. *P ≤ 0.0001 as compared to control; ^#P ≤ 0.005.

Syntaxin 6 is upregulated in Enz-resistant PCa and colocalizes with CD63

After demonstrating that exosomes were critical to the survival of resistant cells, we next wanted to evaluate syntaxin 6 expression in these cell lines based upon its known role in exocytosis. We found that syntaxin 6 was significantly upregulated in the resistant cell lines, as compared to their corresponding sensitive cells (Figure 3A). In order to determine if syntaxin 6 was directly involved in the formation and release of exosomes, we evaluated its colocalization with CD63. CD63 is a tetraspanin protein that is enriched in membrane microdomains and is often used as an exosomal biomarker. CD63 is also present on MVEs containing exosomes within the cells. When comparing the colocalization of syntaxin 6 and CD63 in C4–2B-S and C4–2B-R cells, we found significantly greater colocalization in C4–2B-R (Figure 3B). There was 27.0% more colocalization in the resistant cells as compared to sensitive cells (P ≤0.0001). We then used Mander’s Colocalization Coefficient to analyze the fraction of each protein that colocalizes with the other in both cell lines. We found that while the fraction of CD63 that colocalizes with syntaxin 6 remained similar between C4–2B-S and C4–2B-R (0.498 vs 0.529), but the fraction of syntaxin 6 that colocalizes with CD63 increased significantly in C4–2B-R (0.507) compared to C4–2B-S (0.2931) (P =0.0006).

Figure 3. Syntaxin 6 is upregulated in Enz-resistant PCa cells and colocalizes with CD63.

(A) Immunofluorescence of syntaxin 6 in the Enz-sensitive(S) and Enz-resistant (R) C4-2B, CWR-R1, and LNCaP cell lines analyzed by confocal microscopy. Representative images are shown at 60x. (B) A comparison of the colocalization of syntaxin 6 with CD63 in DAPI (blue) stained sensitive and resistant C4-2B cells (white arrows indicate colocalization). Pearson coefficient was utilized to quantify the colocalization. Mander’s Coefficient was utilized to quantify the proportion of syntaxin 6 that colocalizes with CD63 (M1), as well as the proportion of CD63 that colocalizes with syntaxin 6 (M2). N=5 images each *P ≤0.0001; ‡ P ≤0.001.

Syntaxin 6 is critical for the survival of Enz-resistant PCa cells

We next evaluated whether inhibition of syntaxin 6 expression would lead to decreased cell survival in the presence of Enz in C4–2B-R and CWR-R1-R cell lines. Western blot analysis showed that upon transfection of syntaxin 6 specific siRNA, the level of syntaxin 6 protein was significantly reduced in C4–2B-R cells (Figure 4A). We then performed a trypan blue assay of the knockdown cell lines with and without Enz treatment. In C4–2B-R, syntaxin 6 knockdown led to a significant decrease in the number of living cells, but only when treated with Enz (Figure 4B). Whereas in the absence of Enz, syntaxin 6 knockdown led to an 8.6% decrease in living cells (P =0.463); in the presence of Enz, knockdown led to a 43.9% decrease in living cells (P =0.039). Furthermore, we found that that there was a significant increase in the percentage of dead cells, but once again only after Enz treatment (Figure 4C). In the presence of Enz, syntaxin 6 knockdown led to a 3.5-fold increase in the percentage of dead cells (P ≤0.0001). Exosome secretion was also analyzed in syntaxin 6 knockdown cells, with exosome concentration normalized for cell number. Syntaxin 6 knockdown in C4–2B-R led to a 41.7% (P ≤0.0001) decrease in exosomes concentration (Figure 4D).

Figure 4. Syntaxin 6 is critical for the survival of Enz-resistant PCa cells.

C4-2B-R and CWR-R1-R cells were seeded at a density of 3×10⁵ cells/well in 6-well plates. After 24 hours of transfection, fresh media was exchanged with either DMSO (vehicle) or Enz (20 uM). After 48 hrs, cell number and cell death were counted using the trypan blue exclusion assay. Exosomes were isolated from conditioned media and analyzed for particle size distribution and concentration by NTA as described in methods. (A) Western blot confirming knock-down of Syntaxin 6 in C4-2B-R cells. Densitometry values are presented below the bands. (B-C) Live cell number and cell death in C4-2B-R cells following syntaxin 6 knockdown in the presence or absence of Enz. (D) Exosome concentration after syntaxin 6 knockdown in C4-2B-R cells. (E) Western blot confirming knockdown of Syntaxin 6 in CWR-R1-R cells. Densitometry values are presented below the bands. (F-G) Live cell number and cell death in CWR-R1-R- cells following syntaxin 6 knockdown in the presence or absence of Enz. (H) Exosome concentration after syntaxin 6 knockdown in CWR-R1-R cells. Values are expressed as mean±SEM, n=3. TR: Transfection Reagent alone; ND: Not detectable; Stx6KD: syntaxin 6 knockdown; ‡P ≤ 0.05 *P ≤ 0.0001.

Similarly, transfection of syntaxin 6 specific siRNA almost completely reduced the syntaxin 6 expression in CWR-R1-R cells (Figure 4E), which resulted in a significant decrease in living cells after Enz treatment (P=0.031), with no significant decrease without treatment (P<0.2296, Figure 4F). Whereas in the absence of Enz, syntaxin 6 knockdown led to a 27.2% decrease in living cells; in the presence of Enz, knockdown led to a 61.9% decrease in living cells. There was no significant difference in the percentage of dead cells in the presence of Enz (P=0.135) or in its absence (P=0.741, Figure G). Syntaxin 6 knockdown in CWR-R1-R led to a 69.7% (P ≤0.0001) decrease in exosome concentration (Figure 4H).

Clinical Significance of syntaxin 6 expression in PCa

In order to correlate our in vitro results with clinical data, we queried The Cancer Genome Atlas (TCGA)-prostate adenocarcinoma for syntaxin 6 mRNA expression. We found that increased expression of syntaxin 6 was correlated with higher Gleason score (P = 3.16×10⁻⁸), stage of the primary tumor (P = 9.44×10⁻⁸) as well as decreased progression free survival (P =0.018), (Figure 5 A-C).

Figure 5. Syntaxin 6 expression from the TCGA-PCa and PCBN databases.

Expression of syntaxin 6 was analyzed as a function of (A) Gleason score and (B) pathologic stage in PCa patients from the TCGA dataset. (C) Patient cohort was then stratified into three groups based on syntaxin 6 expression; mean ± 1SD (Low, Medium, and High expression). Kaplan-Meir curve was then generated based on progression free survival. (D) Metastatic PCa tissue from autopsy samples (liver, lung, bone, and lymph node) were assayed for syntaxin 6 protein expression via IHC as detailed in the methods. Immunoreactivity was analyzed throughout each tissue sample and was scored as 0+ (no staining), 1+ (weak staining), 2+ (moderate staining), 3+ (strong staining), and 4+ (very strong staining). The areas of staining score 1 to 4 were measured respectively, then the ratios of the area for each score versus the total area were calculated. The ratios were compared between Enz-treated (n=10) and Enz non-treated patients (n=10) for each scoring intensity. Representative images are shown.

Given the positive correlation of syntaxin 6 with PCa grade and pathologic stage, we developed robust IHC detection of syntaxin 6 protein expression using autopsied metastatic prostate tumors collected by the PCBN. The collection of tissue samples was comprised of 10 Enz-naïve patients and 10 patients who had received Enz. A semi-quantitative immunoreactivity scoring system of 0 to 4 reflecting intensity of staining was used. Those samples from treated patients demonstrated greater positive staining than the non-treated ones. This can be seen at all staining intensities. However, these differences did not achieve statistical significance (Figure 5D).

Discussion

Enz resistance has proven to be a key problem in the treatment of mCRPC. This requires a greater understanding of resistance development in order to improve treatment modalities for patients. In our research, we have identified a unique mechanism of Enz resistance that relies upon the higher secretion of exosomes regulated by syntaxin 6 expression.

Exosomes are emerging as a powerful tool for cancer diagnosis and prognosis. As evidence of this realization, Exosome Diagnostics Inc (exosomedx) has launched several exosomes based tests to aid clinicians in the diagnostic and therapeutic decision making process. The ExoDx Prostate (IntelliScore), for example, is a non-invasive, urine-based test designed to be used in conjunction with PSA to enable clinicians to predict whether a patient presenting for an initial biopsy does not have high-grade PCa.³³ It will be interesting to see whether exosomes could also be useful to predict Enz sensitivity or susceptibility to develop Enz resistance. We observed unique proteins loaded in exosomes secreted by C4–2B-S (210 proteins) and C4–2B-R (42 proteins). However, such analyses need to be performed in multiple cell lines to identify unique biomarkers associated with Enz resistance. Besides, this could be extended to analyses of RNAs, lipids and metabolites loaded in exosomes to identify unique biomarkers associated with Enz resistance development.

Our results suggest that syntaxin 6 could be a key player in the secretion of exosomes as knockdown of syntaxin 6 resulted in a decrease in exosome secretion in Enz resistant PCa cells. The resistance mechanism of drug efflux has commonly been associated with the multidrug-resistant proteins (MDRP), of which include P-glycoprotein and MRP1.³⁴ These proteins belong to the superfamily of ATP-binding cassette (ABC) transports that carry various molecules across extra- and intra-cellular membranes.³⁵ Resistance to docetaxel and other taxanes through these MDRPs have been shown in breast, ovarian, lung, and even prostate cancer.³⁶ However, the structural features of Enz prevent it from being pumped into the extracellular environment through these transporters.³⁷ Therefore, in the absence of a key role for drug efflux pump, we predict that exosome secretion could be a key pathway for Enz drug resistance.

The mechanism through which syntaxin 6 is upregulated and colocalizes with CD63 in Enz resistant cells has yet to be elucidated. A group has previously demonstrated that inhibition of syntaxin 6 leads to a decrease in CD63 expression, preventing release of secretory granules from neutrophils.³⁸ In addition, several studies have demonstrated the regulation of syntaxin 6 by intracellular cholesterol level.^39–41 These studies have shown that Golgi-associated cholesterol is required for syntaxin 6 localization in the trans-Golgi network and for the syntaxin 6-dependent integrin recycling process. Another study found that the protein receptor encoded by Met proto-oncogene is transported to the cell surface via a process that also requires the participation of cholesterol-regulated syntaxin 6 expression.⁴² This is particularly interesting in regards to Enz resistance because a study has shown that in intracellular cholesterol levels are increased in Enz-resistant PCa cells.³⁶ Thus, we propose that increases in cholesterol lead to not only an increased expression of syntaxin 6 but increased co-localization with CD63. It will be important in future studies to understand exactly how cholesterol regulates syntaxin 6 expression in these resistant cell lines. Because these prior studies have illustrated that it is not only the total cholesterol that is important but organelle-specific levels as well, therefore we must determine where exactly the increase in cholesterol within Enz resistant cells is occurring. In turn, it will be necessary to elucidate what is driving these cholesterol changes, and thus what is upstream of syntaxin 6.

Results from present study suggest that Enz-resistant PCa cells could be targeted by exosome biogenesis inhibitors or through syntaxin 6 expression inhibition. Current exosome biogenesis inhibitors used in research are non-specific and target pathways that may be important to normal cellular function. Therefore, it will be important going forward to develop more selective inhibitors. Interestingly, Datta et al. identified the natural metabolite, manumycin-A, as a candidate that inhibits exosome biogenesis and secretion in CRPC cells, but not in normal cells.⁴³ However, a syntaxin 6 inhibitor has yet to be developed. Given our current findings of the role syntaxin 6 plays in Enz-resistant PCa, it is imperative to utilize high-throughput screening and novel drug design to identify a suitable agent.

Overall, in the present study we have identified that the syntaxin 6-mediated exosome secretion regulates Enz resistance in PCa.

Supplementary Material

Supp figS1. Characterization of exosomes isolated from Exo^{Enz-Sensitive} and Exo^{Enz-resistant} proteins by mass spectrometry.

(A) Total proteins (both distinct and overlapping) present in Exo^{Enz-Sensitive} and Exo^{Enz-resistant} are presented in a Venn diagram (Left Panel). Proteins were characterized using Ingenuity IPA software, and proteins subcellular (or extracellular) localization is presented in pie diagrams (Middle and Right Panel). (B) Exo^{Enz-Sensitive} and Exo^{Enz-resistant} total protein data were compared using Ingenuity IPA software. Comparisons of the Top 15 canonical pathways are shown. Scores (total number of proteins in the sample versus total number of known proteins of that pathway) are plotted as –log value, which is derived from a p-value and indicates the likelihood of the Focus Proteins in a network being found together due to random chance. Threshold value is set at 1.25. (C–D) Exo^{Enz-Sensitive} and Exo^{Enz-resistant} proteins clustered within the Top Networks/Associated Functions as derived from IPA algorithms are shown as members of the “interactomes”. Protein shapes are indicative of function and that legend is shown.

Abbreviations:

ABC

ATP-binding cassette

AR

Androgen Receptor

CCV

Clathrin-coated vesicles

DMA

5-(N,N-Dimethyl)amiloride hydrochloride

DMSO

Dimethyl sulfoxide

ECL

Enhanced chemiluminescence

Enz

Enzalutamide

FAK

Focal adhesion kinase

FBS

Fetal bovine serum

IF

Immunofluorescence

IHC

Immunohistochemistry

IPA

Ingenuity pathway analysis

mCRPC

metastatic castration-resistant PCa

MDRP

Multidrug-resistant proteins

MVB

Multivesicular bodies

MVE

Multi-vesicular endosomes

NTA

Nanoparticle tracking analysis

PCa

Prostate cancer

PCBN

Prostate cancer biorepository network

PSA

Prostate-specific antigen

SNARE

Soluble N-ethylmaleimide attachment protein receptor