CANCER GENOMICS & PROTEOMICS 20: 456-468 (2023)
doi:10.21873/cgp.20397

H19 in Serum Extracellular Vesicles Reflects Resistanceto AR Axis-targeted Therapy Among CRPC Patients

TAKU KATO1,2, KYOJIRO KAWAKAMI3, KOSUKE MIZUTANI4, TATSUYA ANDO1, YASUHIRO SAKAI1,
KOUHEI SAKURAI1, SHOHEI TOYOTA2, HIDETOSHI EHARA2, HIROYASU ITO1 and MASAFUMI ITO3
1Department of Joint Research Laboratory of Clinical Medicine,

Fujita Health University School of Medicine, Toyoake, Japan;

2Department of Urology, Asahi University Hospital, Gifu, Japan;

3Research Team for Functional Biogerontology, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan;
4Department of Urology, Central Japan International Medical Center, Gifu, Japan

Abstract. Background/Aim: We aimed to evaluate thechanges of androgen receptor (AR) signaling-related longnon-coding RNAs (lncRNAs) in serum extracellular vesicles(EVs) from prostate cancer (PC) patients, in order to identify
novel biomarkers for AR axis-targeted therapy (ARAT)resistance among castration-resistant PC (CRPC) patients.
Patients and Methods: EVs were isolated from 2 patients
before and after acquiring ARAT-resistance. RNA profilingof EVs was performed by RNA-sequencing. The expression
levels of selected lncRNAs in EVs were analyzed by digital
droplet PCR (ddPCR) in 58 localized and 14 metastatic PC
patients at diagnosis, 7 ARAT-naïve and 6 ARAT-resistant
CRPC patients. LncRNA H19 expression in PC tissue was
examined using published data. In order to analyze the roleof H19, the prognosis was analyzed in PC patients andproteomic analysis was performed in 22Rv1 PC cells.
Results: RNA-sequencing revealed that AR-regulated RNAs
were most enriched in EVs after acquiring ARAT-resistance.
Among them, up-regulation of AR signaling-related lncRNAs(PCAT1, H19, HOXA-11AS, ZEB1-AS1, ARLNC1, PART1,
CTBP1-AS and PCA3) was confirmed by ddPCR. H19
contained in EVs (EV-H19) was significantly increased

among ARAT-resistant patients compared to ARAT-naïve
CRPC or metastatic PC patients. In PC tissue, H19 was
negatively correlated with AR protein and AR-activity scoreand up-regulated in neuroendocrine CRPC tissue with low
AR expression. Furthermore, EV-H19 expression was
significantly associated with worse outcome to androgendeprivation therapy. Proteomic analysis demonstrated thatH19 knockdown enhanced PC-related protein expression.
Conclusion: EV-H19 may negatively correlate with ARsignaling activity and could be a marker to diagnose ARATresistance among CRPC patients.

Prostate cancer (PC) is the most commonly diagnosed malemalignancy in the United States, with 268,490 new cases andthe second leading cause of death with 34,500 deaths in 2022
(1). The five-year overall survival rate among localized andregional PC patients is over 99%, however, and it decreases
to 31% among metastatic PC patients (1). Androgen receptor(AR)-signaling is a central axis in PC pathogenesis andandrogen-deprivation therapy (ADT) is a common treatment
for metastatic PC patients. ADT is temporarily effective;
however, PC develops castration-resistant PC (CRPC)
following ADT. It has been reported that AR-signaling is still
activated in CRPC (2). For this reason, second generation
androgen receptor axis-targeted therapy (ARAT; Abiraterone,
Enzalutamide, Apalutamide and Darolutamide) is strongly
recommended for CRPC patients by the National
Comprehensive Cancer Network (NCCN) guidelines (3).
Although ARAT improves the prognosis of CRPC, it has
limited effect and CRPC patients develop ARAT-resistance(4). Prostate-specific antigen (PSA) is a reliable tumor
monitoring marker for PC; however, it sometimes deviatesfrom the disease state in CRPC patients who are receiving
ARAT (5). Therefore, it is of great importance to identify
novel biomarkers to diagnose ARAT-resistance accurately. AR
mutations and splicing variants are one of the known

Correspondence to: Taku Kato, MD, Ph.D., Department of Joint
Research Laboratory of Clinical Medicine, Fujita Health University
School of Medicine, 1-98 Dengakugakubo, Kutsukake-cho,
Toyoake, Aichi, 470-1192, Japan. Tel: +81 562932000, e-mail:taku.kato.cp@fujita-hu.ac.jp

Key Words: Prostate cancer, CRPC, extracellular vesicles, H19,
RNA-sequencing, androgen receptor axis-targeted therapy.

This article is an open access article distributed under the terms andconditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0

international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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CANCER GENOMICS & PROTEOMICS 20: 456-468 (2023)

mechanisms of ARAT-resistance (4, 6). Pathological evidenceof AR signaling-related gene expression change may support
treatment selection; however, serial biopsy of primary or
metastatic PC site is invasive and not routinely done.
Extracellular vesicles (EVs) are small vesicles that are
secreted from cells to body fluids and contain cellularcomponents such as protein and RNA, thus including the cell
information; they are considered as a promising biomarker
for various diseases including PC (7). Recent transcriptomic
analysis revealed that there was a positive correlation
between RNA expression in EVs (EV-RNA) and that in PC
tissue (8). Del Re et al. reported that AR splice variant 7(AR-V7) mRNA contained in plasma EVs, which lacks theandrogen binding domain and is in part involved in ARATresistance, was a predictive biomarker for ARAT treatment(9). Therefore, EV-RNA can provide PC information
including AR-signaling and ARAT-resistance as a lessinvasive blood-based test.
Long non-coding RNAs (lncRNAs) are non-protein coding
RNAs that are over 200 base pairs. In the past few decades,
high-throughput RNA-sequencing (RNA-seq) revealed that ten
thousand of lncRNAs are present, much more than protein
coding mRNAs (10). LncRNAs have been implicated incancer development such as cell growth, invasion, metastasis,anti-apoptosis, mis-regulated cell cycle, etc. through binding
to proteins or sponging to miRNAs (10). In PC, some
lncRNAs (e.g., PCAT1, PCAT14, MEG3, MALAT1,
HOTAIR) have been reported as potential diagnostic markers
or therapeutic targets (11). As for lncRNAs contained in EVs(EV- lncRNAs), only 5 EV-lncRNAs (PCA3, MALAT1 andlncRNA-21p in urine, SAP30L-AS1 and CChLAP1 inplasma) have been reported as potential diagnostic markers forPC (12-15) and EV-lncRNAs to diagnose ARAT-resistance in
CRPC patients have not yet been identified.
In this study, we aimed to identify novel EV-based AR
signaling-related biomarkers for ARAT-resistance with a
special interest in lncRNAs. We performed RNA-sequencingof EVs isolated from serum of the same CRPC patients
before and after acquiring ARAT-resistance and confirmed
the expression changes of selected lncRNAs by digital
droplet PCR (ddPCR) in a cohort of PC patients. We thenfocused on a particular EV-lncRNA and analyzed its
expression, function and clinical significance.

Table I. Patient characteristics of 2 patients subjected to RNA-seq and
8 selected androgen receptor (AR)-related lncRNAs whose expression
was upregulated after acquiring AR axis-targeted therapy (ARAT)resistance.

Age
PSA (ng/ml)
Initial diagnosis
Before ARATBest response during ARAT
Clinical TNM
Gleason grade
Selected lncRNA*
PCAT1
H19
HOXA11-AS
ZEB1-AS1
ARLNC1
PART1
CTBP1-AS
PCA3

Patient 1

Patient 2

Unknown
17.262
5.981
T4N1M1b
Unknown

580.104
24.79123.035
T4N1M1bGroup 588

15.4631697
14.3842161
14.7709495
15.4958416
11.245032
4.65646381
3.63387392
2.89962403

92

14.86513079
15.04641865
14.53329095
11.80097665
13.94166309
12.10653614
3.141891981
2.973985109*The fold expression change of each lncRNA after acquiring ARATresistance is shown in log2 scale.

patients were used, which included 58 therapy-naïve localized PC(localized PC), 14 therapy-naïve metastatic PC (metastatic PC), 7
ARAT-naïve CRPC (CRPC) and 6 ARAT-resistant CRPC (ARATresistant) patients. Patient characteristics are summarized in Table II.
Two patients, whose serum was used for RNA-seq, were also
included in the confirmation study. To evaluate progression freesurvival, we compared EV-lncRNA expression in 28 patients who
received ADT as a first-line therapy. Serum was centrifuged at 1,500
× g for 10 min to remove cells. Samples were then stored at –80˚Cuntil use. CRPC- or ARAT-resistance was diagnosed by the increasing
PSA level or radiographic progression with castration level of serum
testosterone (<50 ng/dl), respectively.

EV isolation. Highly-purified EVs were prepared by PS-affinity
method as we described previously (16, 17). In this study, we
isolated EVs using MagCapture™ Exosome Isolation Kit PS Ver.
2 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan)
according to the manufacturer’s instructions. Briefly, serum or cell
culture medium was centrifuged at 12,500 × g to remove large
microvesicles. The supernatant was incubated for 1 h at room
temperature with Tim4-conjugated beads. EV-captured beads weredirectly used for subsequent experiments without releasing EVs.

Patients and Methods

Patients. This study was approved by the ethics committee of Fujita
Health University (# HM22-032), Asahi University Hospital (# 201910-06) and Central Japan International Medical Center (# 2022-0122). Written informed consent was obtained from all patients. Serumwas collected from 2 CRPC patients before ARAT-administration andafter acquiring ARAT-resistance for RNA-seq to compare EV-RNA
expression (Table I). To confirm EV-RNA expression changes, sera
collected between January 2020 and October 2022 from 85 PC

457

Total RNA extraction and complementary DNA synthesis. Total
RNA was extracted using the miRNeasy mini kit (Qiagen, Valencia,
CA, USA) according to the manufacturer’s protocol. EV-RNA
isolated from 1,000 μl of serum was used for RNA-seq and thatfrom 500 μl of serum or cell culture medium was used to confirm
RNA expression by reverse transcription (RT)-ddPCR or
quantitative RT-PCR (qRT-PCR). Complementary DNA (cDNA)
synthesis was performed using the iScript cDNA synthesis kit (BIORAD, Hercules, CA, USA). For RT-ddPCR, 15 out of 20 μl of thetotal volume containing total RNA isolated from serum EVs wasKato et al: EV-H19 as a Maker for ARAT-resistance
Table II. Patient characteristics.
Number of cases
Age
Initial PSA (ng/ml)
Clinical T
T1
T2
T3
T4
TX
Clinical N
N0
N1
NX
Clinical M
M0
M1
MXGleason grade
1
2
3
4
5
Unknown

Localized PC

Metastatic PC

CRPC

ARAT-resistant58
73 (58-88)
10.08 (3.90-475.86)

1477 (66-95)
128.94 (0.81-1298.49)

7
88 (66-92)
143.57 (32.82-601.20)

6
81 (64-92)
192.52 (15.59-580.10)

58 (100%)
0 (0%)
0 (0%)

9 (64.3%)
5 (35.7%)
0 (0%)

3 (42.6%)
3 (42.6%)
1 (14.3%)

3 (50%)
2 (33.3%)
1 (16.7%)

8 (13.8%)
28 (48.3%)
18 (31.0%)
4 (6.9%)0 (0%)

58 (100%)
0 (0%)
0 (0%)

34 (58.6%)
9 (15.5%)
1 (1.7%)8 (13.8%)5 (8.6%)
1 (1.7%)1 (7.1%)
3 (21.4%)
6 (42.9%)
4 (28.6%)
0 (0%)

0 (0%)
14 (100%)
0 (0%)

0 (0%)
1 (14.3%)
1 (14.3%)
4 (57.1%)
1 (14.3%)

1 (14.3%)
5 (71.4%)
1 (14.3%)

0 (0%)
1 (7.1%)2 (14.3%)
4 (28.6%)
4 (28.6%)
3 (21.4%)

1 (14.3%)
1 (14.3%)
0 (0%)
0 (0%)
2 (28.6%)
3 (42.6%)

0 (0%)
1 (16.7%)
2 (33.3%)
2 (33.3%)
1 (16.7%)

Median (range)
Median (range)

1 (16.7%)
4 (66.7%)
1 (16.7%)

1 (16.7%)
0 (0%)
1 (16.7%)
0 (0%)
2 (33.3%)
2 (33.3%)

Cases (%)

PC, Prostate cancer; CRPC, castration-resistant prostate cancer; AR, androgen receptor; ARAT, androgen receptor axis-targeted therapy.

subjected to cDNA synthesis. For qRT-PCR, 500 ng of RNA from
cells or 10 ng of EV-RNA from cell culture medium was subjected
to cDNA synthesis after determination of RNA concentration.
cDNA was stored at –20˚C until use.

AR-V7; AIQJDUH, PCAT1; Hs04275386_s1, H19; Hs00399294_g1,
HOXA11-AS; Hs03296531_m1, ZEB1-AS; Hs01398645_g1,
ARLNC1; Hs01596342_m1, PART1; Hs00213199_m1, CTBP1-AS;
Hs04401800_m1, PCA3; Hs01371939_g1, GAPDH; Hs03929097_g1).
RT-ddPCR was performed using QX200 AutoDG Droplet Digital PCR
system (BIO-RAD), where 1.1 μl of cDNA was used to generate each
droplet. The following conditions were used for PCR: 95 ˚C × 10 min,
94 ˚C × 30 s, 60 ˚C × 1 min (60 cycles), 98 ˚C × 10 min, 10 ˚C hold.
The fluorescence signal of the PCR reaction was measured using adroplet reader. Absolute quantification was determined using QuantaSoft
software (BIO-RAD). Then, RNA copy number contained in EVs wascalculated. Since a normalization method for EV-RNA has not yet been
established, the expression level of each RNA in EVs was represented
as EV-RNA copies in 1 ml of serum (copies/ml) in this study.

RNA-sequencing. RNA-seq was conducted at the TAKARA Bio
incorporation (Kusatsu, Japan). Briefly, cDNA was synthesizedusing random primer and a specific sequence was assigned to theend of the 1st strand cDNA by the SMART method (18). Then,
using the specific sequences, samples were amplified by PCR using
indexed primers having different tag sequences for each sample.
The obtained PCR product was purified by the magnetic beadmethod using AMPure XP (Beckman Coulter, Brea, CA, USA), andthe ribosomal cDNA was cleaved. The uncleaved cDNA was PCRamplified using adapter-specific primers to form a sequencing
library. The library quality was determined by Agilent 2100
BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA). The
strand-specific RNA-seq libraries were sequenced using theNovaSeq 6000 platform (Illumina, San Diego, CA, USA). The
results of RNA-seq were analyzed using iDEP.96 (http://bioinformatics.sdstate.edu/idep96/) and Metascape software (https://
metascape.org/gp/index.html#/main/step1). Based on the result ofRNA-seq, 8 AR signaling-related lncRNAs were selected to confirm
expression changes (Table I).

Database analysis. Databases of The Cancer Genome Atlas (TCGA)
and Weill Cornel Medicine (WCM) were downloaded fromcBioPortal for cancer genomics (https://www.cbioportal.org). Basedon the database of TCGA, lncRNA H19 expression was comparedto AR protein and AR activity score determined by the expression
pattern of 20 AR transcriptional targets (19). We used WCM cohort
to compare AR mRNA and H19 expression among CRPC specimens
with adenocarcinoma phenotype (CRPC-Adeno) and neuroendocrine (NE) phenotype (CRPC-NE).
Cell culture. Human CRPC C4-2B cells were obtained from the MD
Anderson Cancer Center (Houston, TX, USA) and human PC
22Rv1 cells were from the European Collection of AuthenticatedDroplet digital PCR. To determine the expression levels of AR and ARV7 mRNA as well as 8 lncRNAs, Taqman probes were purchased from
Thermo Fisher Scientific (Waltham, MA, USA, AR; Hs00171172_m1,

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Figure 1. Transcriptomic analysis before and after acquiring androgen receptor axis-targeted therapy (ARAT) resistance. (A) Heatmap of RNA-seq
among 2 castration-resistant prostate cancer (CRPC) patients before and after acquiring ARAT-resistance. A total of 9323 genes are identified. (B)Upstream transcription factors that up-regulated transcripts by 2 folds or more in 2 patients after acquiring ARAT-resistance are listed in descendingorder of the number of transcripts each transcription factor regulated.

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Kato et al: EV-H19 as a Maker for ARAT-resistance

Figure 2. Androgen receptor (AR)-related lncRNAs expression among prostate cancer (PC) patients evaluated by reverse transcription-droplet digital
PCR (RT-ddPCR). EV-H19 was significantly up-regulated in ARAT-resistant PC patients compared with metastatic or CRPC patients (median 100
copies/ml vs. 0 and 0, p=0.0013 and 0.0104). *p<0.05, **p<0.005, #p<0.10 using the Kruskal-Wallis test with Dunn’s multiple comparison test.

Cell Cultures (ECACC, Salisbury, UK). Cells were cultured in
RPMI 1640 medium supplemented with 10 % fetal bovine serum(FBS), 100 units/ml of penicillin and 100 mg/ml of streptomycin ina humidified atmosphere containing 5 % CO2.

Proteome Discoverer 2.4 software (Thermo Fisher Scientific).
Metascape software was used for enrichment analysis.

Small interfering RNA transfection. Two small interfering RNA(siRNA) for H19 and negative control siRNA were purchased from
Dharmacon (Lafayette, CO, USA). The target sequences of thesiRNAs were as follows: H19 siRNA1 (GGACGAUGGG
CCUGAGCUA); H19 siRNA2 (GGAGCAGCCUUCAAGCAUU);
negative control siRNA (UAGCGACUAAACACAUCA). Cells
seeded onto 6-well plates and left for 24 h were transfected withsiRNA for 4 h at 10 nM using the Lipofectamine™ RNAiMAX
transfection reagent (Thermo Fisher Scientific), according to the
manufacturer’s instructions. 48 h after transfection, total RNA wasextracted in triplicate from cells and EVs were isolated from cell
culture medium.

Results

Quantitative reverse transcription-PCR. To determine the H19
expression levels in cells, qRT-PCR was performed using Taqman
fast advanced master mix (BIO-RAD). The following conditions
were used for PCR: 50 ˚C × 2 min, 95 ˚C × 20 s, 95 ˚C × 1 s and
60 ˚C × 20 s (50 cycles), 4 ˚C hold. Cellular H19 was normalized
to GAPDH mRNA, and a relative fold change was calculated using
the comparative Ct method.

Statistical analysis. The student t-test was used to compare twoparametric groups and Mann-Whitney U-test for non-parametricgroups. One-way ANOVA with Fischer’s LSD was used to compare
three parametric groups and Kruskal-Wallis test with Dunn’s multiple
comparison test was used for three or more non-parametric groups.
Kaplan Meier method and generalized Wilcoxon test were used toevaluate survival. The data analysis was performed using GraphPad
Prism 9 software (San Diego, CA, USA). p<0.05 was considered asstatistically significant and p<0.10 as a statistically significant trend.

Nano liquid chromatography tandem mass spectrometry (nanoLCMS/MS). Proteins were extracted using RIPA buffer 48 h after
siRNA transfection in triplicate. Trypsin digestion prior to proteomic
analysis was performed using S-Trap micro columns (ProtiFi, NY,
USA) according to the instruction manual. Desalting of peptides
after trypsin digestion was conducted using styrene-divinylbenzene(SDB) stage-Tip. Proteomic analysis was performed as described
previously (17). All data were analyzed for protein identification by

RNA-sequencing and ddPCR revealed that H19 was upregulated in ARAT-resistant patients. Firstly, we isolated EVRNA from the serum of 2 CRPC patients before and after
acquiring ARAT-resistance, who were initially sensitive to
ARAT, and then performed RNA-seq. The results showed that2958 RNAs were up-regulated and 16 were down-regulated,
respectively, by 2-fold or more after acquiring ARAT-resistancecompared with ARAT-naïve (Figure 1A). Among up-regulatedtranscripts, those regulated by AR were the most enriched, whenanalyzed
bythe
TRRUST
database(http://www.grnpedia.org/trrust) (Figure 1B). We then selected 8
AR-related lncRNAs from the up-regulated lncRNAs to confirm
their expression changes using ddPCR (Table I). The H19 copy
number was significantly elevated in ARAT-resistant PC patientscompared to metastatic PC and CRPC patients (Figure 2). The
expression levels of PCAT1 and HOXA11-AS were not changed

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Figure 3. Correlation between H19 and androgen receptor (AR) expression in prostate cancer (PC) tissue. The correlation of H19 expression with
AR protein (A) expression and AR activity score (B) in PC tissue is represented. Data were downloaded from The Cancer Genome Atlas (TCGA)
database (n=333). (C) AR mRNA and H19 expression among 34 CRPC-Adeno and 15 CRPC-NE specimens from the Weil Cornel Medicine (WCM)
cohort (p<0.0001 and p=0.0009). RNA expression is shown in log2 scale. ***p<0.001 and ****<0.0001 using the Mann-Whitney U-test.

among these patients. AR, AR-V7 mRNA and other lncRNAs
were not detected in these samples (data not shown).

EV-H19 expression associates with worth outcome among
patients who received androgen-deprivation therapy. To
evaluate the association between EV-H19 expression and PC
progression, we performed survival analysis among 28 PC
patients who received ADT as a first-line therapy. We foundthat EV-H19 was positive in 11 patients and that thosepositive for EV-H19 had a significantly higher probability of
disease progression (Figure 4).

H19 is negatively correlated with AR and up-regulated inneuroendocrine CRPC among PC tissues. To evaluate thecorrelation between H19 and AR expression in PC tissue, we
compared the H19 expression with AR protein expression
and AR activity score using the TCGA cohort. We found avery weak but negative correlation between H19 and AR
protein expression and AR activity score in PC tissue (Figure
3A and B). We also compared AR mRNA and H19
expression among CRPC-Adeno and CRPC-NE tissues using
the WCM cohort. AR mRNA and H19 were significantly
down-regulated and up-regulated, respectively, in CRPC-NE
compared to CRPC-Adeno specimens (Figure 3C).

EV-H19 correlates with cellular H19 expression and H19knockdown enhances the PC phenotype. The 22Rv1 CRPC
cells express AR-V7 protein and share some features withNE cells (20, 21). On the other hand, C4-2B cells express
AR wild-type protein with low AR-V7 protein expression
and are considered to be typical CRPC cells (20, 22). We
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Kato et al: EV-H19 as a Maker for ARAT-resistancesuch as PI3K/Akt, MAPK, Wnt, JAK/STAT and NF-κB
signaling (24). In colorectal cancer, H19 expression was
significantly higher in cancerous tissue compared to tumor-freesurrounding tissue (25). H19 expression in endocrine tumors is
controversial. High H19 expression was observed in malignant
pancreatic neuroendocrine neoplasms and their liver metastasis
but was found to be low in non-malignant tumors (26). In PC,
Singh et al. demonstrated that AR suppressed H19 transcription
and that H19 was up-regulated in CRPC-NE specimens
compared to CRPC-Adeno (27). CRPC-NE is a lethal
phenotype of PC associated with relative resistance to ARdirected therapies and is characterized by negative expressionof AR and positive expression of NE markers such as neuronspecific enolase (NSE), chromogranin A (CHGA) andsynaptophysin (SYP) (21, 27-30). We confirmed that H19
expression was negatively correlated with AR protein and AR
activity score in PC specimens (Figure 3A and B). Moreover,
AR mRNA and H19 were significantly down-regulated and upregulated, respectively, in CRPC-NE compared to CRPCAdeno specimens (Figure 3C), suggesting that higher tissue
H19 expression may represent lower AR-signaling activity in
PC specimens such as CRPC-NE.
In serum EVs, it has been shown that H19 wassignificantly increased in bladder, gastric and breast cancer
patients compared to control patients and decreased after
tumor resection (31-33). Wang et al. also reported that EVH19 expression was significantly correlated with H19
expression in bladder cancer specimens (31), suggesting that
EV-H19 may be associated with tissue H19 expression incancer. In this study, a similar correlation was observed for
H19 expression between PC cells and EVs (Figure 5A andB), supporting the notion that EV-H19 may also reflect PC
tissue information. Interestingly, EV-H19 was up-regulated
in localized PC patients at diagnosis compared to treatmentnaïve metastatic PC patients (p=0.0860, Figure 2A). It is also
well known that the AR gene is amplified in advanced PC
tissue compared to the primary site and that AR protein is
increased in advanced PC (34, 35). These findings suggestthat higher EV-H19 may indicate lower AR-signaling activity
in PC tissue.
A previous report demonstrated that higher H19
expression in PC tissue was associated with lower
progression free survival among patients who received ADT(27). In this study, we also found that EV-H19 expression
was associated with worse outcome among patients treatedwith ADT (Figure 4), suggesting that H19 expression in PC
tissue and EVs may explain insensitivity to ADT caused by
reduced AR-signaling.
It has also been reported that H19 attenuated AR-signaling
without changing AR protein expression, up-regulated NErelated gene expression through H19-bound PRC2 protein
complex-mediated methylation and changed the phenotype
from CRPC-Adeno to CRPC-NE (27). Several studies

Figure 4. Progression free survival of prostate cancer (PC) patients who
received androgen deprivation therapy (ADT) as a first-line therapy. Red
and blue lines indicate EV-H19 positive (n=11) and negative (n=17)
patients, respectively. Median progression free survival was 15.5 months
in EV-H19 positive patients and undefined in negative patients (p=0.0498
using the Kaplan Meier method and the generalized Wilcoxon test).

compared H19 expression among these two lines. H19 wassignificantly higher in cells and EVs of 22Rv1 cells than inthose of C4-2B CRPC cells (Figure 5A). After H19
knockdown in 22Rv1 cells, EV-H19 was also significantly
reduced (Figure 5B). We then performed proteomic analysis
to investigate H19 function. 114 proteins were significantly
up-regulated and 87 significantly down-regulated after H19
knockdown using both siRNAs, respectively (Table III).
Among up-regulated proteins, those related to “Prostatic
Diseases” and “Metastasis from malignant tumor of prostate”
were enriched, when analyzed using the DisGeNET database(https://www.disgenet.org) (Figure 5C and D). H19
knockdown did not affect AR protein expression (Table III).

Discussion

In the present study, we aimed to identify a novel EV-based
biomarker to indicate AR-signaling activity and to diagnose
ARAT-resistance. As the result of RNA-seq before and after
acquiring ARAT-resistance in the same patients, a quite
different expression pattern of transcripts contained in EVs
was observed (Figure 1). Among up-regulated lncRNAs, we
confirmed that EV-H19 was elevated in ARAT-resistant
CRPC patients compared to localized, metastatic and CRPC
patients (p=0.0735, 0.0013 and 0.0104, Figure 2).
H19 is the first discovered lncRNA with 2.3 kb total length
which is highly expressed in most tissues of the embryogenesis
or placenta and declines postnatally (23, 24). In adult tissue,
H19 is present in skeletal muscle, cartilage and cardiac muscle(24). H19 has also been reported to be expressed in various
types of cancer and regulate oncogenic signaling pathways
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CANCER GENOMICS & PROTEOMICS 20: 456-468 (2023)

Figure 5. Effects of H19 knockdown on EV-H19 expression and proteomic changes in 22Rv1 cells. (A) Cells (2.0×105) cultured in serum-free medium
were seeded onto 6-well plates. 72 h after seeding, total RNA was extracted from the cells and EVs were isolated from the cell culture medium. H19
was significantly higher in cells and EVs of 22Rv1 cells compared to those of C4-2B cells (n=3, 21.01- and 2.37-fold, p=0.0006 and 0.0323, respectively,
student-t test). (B) Total RNA was extracted 48 h after transfection of siRNAs. H19 knockdown in 22Rv1 cells reduced EV-H19 expression (n=3, negative
control vs. siRNA1; 0.239-fold, p=0.0144, negative control vs. siRNA2; 0.266-fold, p=0.0170, one-way ANOVA with Fischer’s LSD). (C) Up-regulated
proteins after siRNA-mediated H19 knockdown were analyzed using the DisGeNET database, in terms of association with diseases. (D) Proteinsregulated by AR are shown in blue letters and those activating AR are in red letters. *p<0.05, ***p<0.001 and ****p<0.0001.

demonstrated that repeated ARAT treatment may lead to aphenotypic change from CRPC-Adeno to CRPC-NE (4, 5,
21, 30). In this study, EV-H19 was significantly elevated in
ARAT-resistant patients compared to metastatic or CRPC
patients (Figure 2). Furthermore, H19 expression, both incells and EVs, was up-regulated in NE-like CRPC 22Rv1
cells compared to C4-2B CRPC cells (Figure 5A). Thesefindings suggest that EV-H19 up-regulation may be
indicative of NE differentiation and could be a marker todiagnose ARAT-resistance among CRPC patients. The
NCCN guideline recommends platinum- or taxane-based
chemotherapy for CRPC-NE patients. EV-H19 expression
could also help select treatment options.

Lastly, we evaluated the effect of H19 knockdown on
protein expression in 22Rv1 cells to investigate H19
function. Although the expression level of AR protein was
not changed, prostate cancer-related proteins were enrichedin up-regulated proteins (Table III, Figure 5C and D). Amongthem, EGFR and FOLH1 were found to be regulated by AR
according to the TRRUST database (Figure 5D, blue letter).
EGFR has been reported as not only an AR-regulated protein
but also as an activator of AR through PI3K-Akt and MAPK
signaling (36, 37). FOLH1 protein is also known as aprostate-specific membrane antigen (PSMA). PSMA isstrongly expressed on the cell surface membrane of prostateadenocarcinoma, whose expression is found in 92% of463

Kato et al: EV-H19 as a Maker for ARAT-resistance
Table III. List of proteins that significantly changed expression after H19 knockdown.
Up-regulated proteins
Gene symbol
WWP2
ASAP2
SKIV2LBAG4PTRHD1
BRSK2
CWC22
KRT17
NDRG4
FTL
TNC
BASP1
BCOR
PYURF
PARD6B
FTSJ3
TRPT1
GMPR
BRWD1
SREK1
CROT
MDK
TMEM65
KRT16
VTI1A
USP24
BCAM
SDC1
DHRS2
DDR1
TANK
VGF
DIP2A
PRKCH
SLC50A1
H1-0
FAM234A
TGFB1
LINC00336
KRT77
SLAIN2
TM7SF2DYNLL2
NIN
C1QL4
EGFR
SLC25A22
FYCO1
RNMT
MAP1B
SPP2
LGALS1
FLOT2
FLG2

Fold change

siH19-1
100
100
100
100
100
100
100
4.188
4.169
3.539
3.355
3.196
2.984
2.974
2.925
2.729
2.641
2.584
2.567
2.458
2.392
2.391
2.384
2.282
2.263
2.256
2.175
2.098
2.035
2.029
1.988
1.977
1.974
1.968
1.955
1.952
1.951
1.883
1.877
1.873
1.836
1.799
1.79
1.749
1.695
1.66
1.641
1.637
1.636
1.635
1.625
1.621
1.596
1.595

siH19-2

100
100
100
100
100
100
100
10.074
2.679
4.547
1.465
1.341
4.434
3.081
2.481
2.26
1.872
1.649
3.378
1.996
1.326
1.979
1.462
12.712
1.481
1.808
1.489
1.387
1.918
1.749
1.813
1.121
1.723
1.303
1.517
1.579
1.663
1.641
3.254
1.406
1.591
1.229
1.945
1.247
1.455
1.277
1.407
1.508
1.314
1.491
1.37
1.226
1.193
1.712

siH19-1<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001
0.00010.0005<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001
0.0002<0.0001<0.0001<0.0001<0.0001
0.00050.0026<0.0001
0.00370.00010.0002<0.0001
0.0121<0.00010.0207<0.0001<0.0001<0.0001<0.0001
0.00010.00010.00030.00010.00550.0004<0.0001
0.0075<0.0001
0.0002
0.011

Down-regulated proteins
Gene symbol

p-ValuesiH19-2<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001
0.00170.0217<0.0001<0.0001<0.0001<0.0001
0.00070.0069<0.0001<0.0001
0.0006<0.0001
0.0034<0.0001
0.03180.0005<0.0001
0.013<0.0001
0.00110.0006
0.04840.002
0.04340.0067<0.0001
0.006<0.0001<0.0001<0.0001
0.00010.0317<0.0001
0.0178<0.0001
0.0334<0.0001
0.00150.0025<0.0001
0.01570.00070.04220.0001

CALU
KRT31
URB2
ACYP2
BNIP1
FCHO2
MACO1
ZNFX1
CLCN4
RIOK3
B3GLCT
PFDN6
ALB
YRDC
SLC7A5
POLR3DASNS
SLC3A2
GCNT4
GPR22
AARS1
ATN1
TBC1D4GPN1
SLC33A1
KLK11
CHMP4CMTHFD2
OGFOD1FLVCR1TAGLN2
HMMR
KIF2C
KPNA2
TSPYL2GOLGA4
BAG1
SEC24DCMSS1
ALDH1L2PSPH
UBE2S
METTL17
MUC13
TSPAN8SLC7A1HMGCS1
PTTG1IP
RTN4IP1
MRPL33STOM
MVD
TSPAN3
DOLK

464

Fold change

siH19-1
0.01
0.01
0.01
0.01
0.01
0.01
0.11
0.133
0.33
0.379
0.418
0.442
0.491
0.495
0.498
0.506
0.525
0.525
0.533
0.545
0.549
0.566
0.569
0.569
0.585
0.587
0.612
0.615
0.619
0.622
0.629
0.644
0.65
0.655
0.656
0.663
0.666
0.67
0.673
0.676
0.676
0.679
0.682
0.684
0.69
0.69
0.692
0.694
0.696
0.698
0.701
0.702
0.708
0.711

siH19-2
0.01
0.01
0.01
0.01
0.01
0.01
0.875
0.01
0.446
0.01
0.6
0.807
0.868
0.827
0.748
0.743
0.639
0.711
0.491
0.594
0.86
0.494
0.736
0.782
0.805
0.488
0.611
0.559
0.705
0.76
0.851
0.695
0.705
0.835
0.768
0.729
0.742
0.532
0.809
0.733
0.75
0.793
0.81
0.61
0.771
0.822
0.789
0.833
0.855
0.729
0.76
0.805
0.752
0.68

siH19-1<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001
0.0003<0.0001<0.0001<0.0001
0.0005<0.0001
0.00640.01820.00030.0008<0.0001
0.0461<0.0001
0.00010.0013<0.0001
0.01160.0398<0.0001
0.04930.00910.00130.01260.0095<0.0001
0.00030.00030.01640.01940.00080.001<0.0001
0.00070.00210.02360.00040.00120.03510.0288

p-ValuesiH19-2<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001
0.0393<0.0001<0.0001<0.0001
0.00010.01140.03570.01730.00020.0299<0.0001<0.0001<0.0001
0.00030.0246<0.0001
0.03510.01890.0335<0.0001
0.0017<0.0001<0.0001
0.00730.01540.00730.01460.00790.04960.00260.0028<0.0001
0.0336<0.0001
0.00020.002
0.0407<0.0001
0.00050.02620.00030.01610.04580.00260.00010.00230.01970.0002

Table III. Continued

CANCER GENOMICS & PROTEOMICS 20: 456-468 (2023)
Table III. Continued
S100A13
COX7A2PVR
LRRC8A
PLPP1
CD63
PREX2
PRPF4B
UBXN7
H1-5
RAMAC
PLBD2
LYPLA2
TUSC3
TRA2A
PHIP
TRA2B
KDELR2F11R
EPS8L2CCZ1B
CLASP1
COLEC12
RNPS1
MAVS
FXYD3
LSM3
MYO1C
BTF3L4
NME3
APLP2
H1-3
NUCKS1YTHDC1RPL18
SEPTIN8
HP1BP3GLG1PCYOX1
L1RE1
ACIN1
MLEC
DDX23
HK1
CAPN1
RPL29
SNRNP70
IQGAP1
FOLH1
PNN
DERPC
HNRNPLSAFBPSME1HNRNPDLALDH6A1
SRSF7
SRSF9
ARPC3
LMNB1

siH19-1
1.585
1.572
1.568
1.553
1.549
1.534
1.523
1.521
1.516
1.512
1.493
1.488
1.486
1.48
1.469
1.447
1.437
1.437
1.405
1.404
1.397
1.394
1.384
1.382
1.378
1.374
1.37
1.367
1.354
1.351
1.351
1.338
1.337
1.337
1.337
1.335
1.334
1.333
1.328
1.327
1.327
1.318
1.317
1.316
1.291
1.284
1.282
1.282
1.276
1.275
1.274
1.271
1.266
1.249
1.248
1.248
1.24
1.238
1.233
1.229

siH19-2
1.366
1.65
1.282
1.505
1.408
1.165
1.832
1.548
1.347
1.558
1.274
1.299
1.329
1.247
1.341
1.673
1.397
1.247
1.3
1.211
1.423
1.25
1.192
1.304
1.25
1.226
1.515
1.175
1.189
1.335
1.219
1.332
1.288
1.255
1.168
1.269
1.234
1.264
1.174
1.425
1.26
1.32
1.405
1.207
1.169
1.157
1.212
1.173
1.166
1.308
1.192
1.208
1.163
1.121
1.335
1.181
1.173
1.201
1.16
1.144

siH19-1

0.00020.00180.00390.015
0.0003<0.0001
0.005
0.01930.0014<0.0001
0.00660.00390.00270.00310.00590.01810.00160.00480.011
0.00730.02550.02540.01850.01710.01680.02530.036
0.00060.01320.02280.03350.00230.01650.04470.00240.03870.00260.03
0.01650.03050.00690.00740.03390.00270.00660.00580.01430.01030.01090.046
0.04160.01330.01130.02270.04050.01740.02790.027
0.03140.0357

siH19-20.0001<0.0001
0.04680.0016<0.0001
0.0264<0.0001
0.00040.0005<0.0001
0.04330.00320.00040.01110.0007<0.0001<0.0001
0.01140.001
0.03460.00020.0211
0.04250.00110.00840.0338<0.0001
0.01710.03040.00020.027<0.0001
0.001
0.00790.00830.00470.00040.00760.0424<0.0001
0.00170.00010.00010.00580.02030.02260.00670.02240.02670.00060.03060.00150.02570.04810.00010.00940.00690.00740.033
0.0215

PIR
CLDN7
GFER
PBK
CDK7
D2HGDH
OTUB2
WDR17
OAT
SLC38A1
RAB21
SARS1
KRT83
CBS
GARS1
PCK2
YARS1
FABP5
IDI1
MYO5A
MTHFD1L
PRSS1
SCD
PPID
PFAS
PLCH1
TARS1
UGDH
PHGDH
SNAP29
PSAT1
CARS1
CPNE3

465

siH19-1
0.712
0.716
0.717
0.718
0.719
0.727
0.73
0.736
0.736
0.754
0.762
0.763
0.764
0.767
0.769
0.771
0.773
0.778
0.781
0.784
0.795
0.798
0.803
0.812
0.814
0.816
0.824
0.834
0.837
0.838
0.839
0.844
0.849

siH19-2
0.859
0.778
0.799
0.861
0.776
0.818
0.634
0.703
0.767
0.778
0.865
0.809
0.828
0.803
0.834
0.809
0.821
0.863
0.871
0.77
0.818
0.728
0.763
0.854
0.85
0.799
0.867
0.858
0.787
0.841
0.793
0.855
0.76

siH19-1

0.002
0.04210.0369<0.0001
0.04180.02090.00660.00770.00090.019
0.004
0.00050.01590.00590.00180.00290.00220.001
0.00130.01890.04090.00690.01270.04250.03140.04460.01310.02710.02950.0446
0.03180.04740.032

siH19-2

0.03170.038
0.02680.02930.04340.0495<0.0001<0.0001
0.00030.00750.04530.00170.01770.00220.006
0.00210.00270.03150.03930.00030.0043<0.0001
0.00020.02940.02290.00190.03280.02160.00020.01580.00040.0231<0.0001

Kato et al: EV-H19 as a Maker for ARAT-resistancemetastatic prostate adenocarcinoma but lost in metastatic NE(38). Sommer et al. reported that AR inhibition using ADTor ARAT induced up-regulation of PSMA protein in LNCaPand C4-2 PC cells with mutant AR (T877A), but not in
LAPC4 PC cells with wild-type AR (39). They also showedthat PSMA protein levels were up-regulated in PC tissues
under ADT compared with treatment-naive PC tissues fromthe same patients. Radiolabeled ligands of PSMA are
currently used in positron emission tomography for the
diagnosis of PC (68Ga-PSMA-PET) and in radioligand
therapy for CRPC patients (177Lu-PSMA-RLT) (40). Thus,
PSMA strongly correlates with AR-signaling and PC
phenotypes and is a key diagnostic and therapeutic moleculartarget for prostate adenocarcinoma. On the other hand,
TGFB1, also up-regulated after H19 knockdown (Figure 5D,
red letter), has been reported to enhance AR-mediatedtransactivation via Smad activation without changing AR
expression (22, 41). Shiota et al. demonstrated that a higher
Gleason score and a worse outcome to ADT were observedin metastatic PC patients with TGFB1 polymorphisms(CT/TT on rs224176 and CA/AA on rs4803455) (42),suggesting that TGFB1 expression may affect the prognosis
of PC patients through AR activation. These findings suggestthat H19 knockdown enhances AR-signaling activity withoutchanging AR protein expression and that H19 may regulate
PC plasticity.

Conclusion

In summary, RNA-seq revealed that AR-regulated genes
were enriched in EVs after acquiring ARAT-resistance.
Among them, lncRNA H19 was up-regulated in EVs isolated
from serum of ARAT-resistant CRPC patients. In PC tissue,
H19 was negatively correlated with AR-signaling activity.
EV-H19 expression was also associated with worse outcome
among PC patients. Furthermore, EV-H19 correlated withcellular H19 expression. Our findings suggest that EV-H19
may negatively reflect AR-signaling activity in PC tissue andcould be a novel biomarker to diagnose ARAT-resistanceamong CRPC patients.

Conflicts of Interest

The Authors declare that there are no conflicts of interest in thisstudy.

Authors’ Contributions

TK designed the study and experiments. TK, ST, and HE recruitedpatients. TK performed and analyzed the experiments. KK
performed proteomic analysis. TK, KK, KM, TA, YS, HI, and MI
participated in planning the study. The first draft of the manuscript
was written by TK and MI. All Authors read, commented on, andapproved the manuscript.

466

Acknowledgements

We would like to thank Dr. Hiroyuki Tezuka, who is affiliated with
the Open Facility Center at Fujita Health University, for his
contribution to the cell culture experiment. This study was
supported by JSPS KAKENHI Grant Number 21K09437,
21K09340, 23K08790 and research fund of Asahi University.

References

1 Siegel RL, Miller KD, Fuchs HE, Jemal A: Cancer statistics, 2022.
CA Cancer J Clin 72(1): 7-33, 2022. DOI: 10.3322/caac.21708
2 Scher HI, Sawyers CL: Biology of progressive, castrationresistant prostate cancer: Directed therapies targeting theandrogen-receptor signaling axis. J Clin Oncol 23(32): 82538261, 2005. DOI: 10.1200/JCO.2005.03.4777
3 Schaeffer EM, Srinivas S, Adra N, An Y, Barocas D, Bitting R,
Bryce A, Chapin B, Cheng HH, D’Amico AV, Desai N, Dorff T,
Eastham JA, Farrington TA, Gao X, Gupta S, Guzzo T, Ippolito
JE, Kuettel MR, Lang JM, Lotan T, McKay RR, Morgan T, Netto
G, Pow-Sang JM, Reiter R, Roach M, Robin T, Rosenfeld S,
Shabsigh A, Spratt D, Teply BA, Tward J, Valicenti R, Wong JK,
Berardi RA, Shead DA, Freedman-Cass DA: NCCN guidelines(R)insights: Prostate cancer, version 1.2023. J Natl Compr Canc Netw
20(12): 1288-1298, 2022. DOI: 10.6004/jnccn.2022.0063
4 Chen Y, Zhou Q, Hankey W, Fang X, Yuan F: Second generation
androgen receptor antagonists and challenges in prostate cancer
treatment. Cell Death Dis 13(7): 632, 2022. DOI: 10.1038/
s41419-022-05084-1
5 Zhao SG, Sperger JM, Schehr JL, McKay RR, Emamekhoo H,
Singh A, Schultz ZD, Bade RM, Stahlfeld CN, Gilsdorf CS,
Hernandez CI, Wolfe SK, Mayberry RD, Krause HM, Bootsma M,
Helzer KT, Rydzewski N, Bakhtiar H, Shi Y, Blitzer G,
Kyriakopoulos CE, Kosoff D, Wei XX, Floberg J, Sethakorn N,
Sharifi M, Harari PM, Huang W, Beltran H, Choueiri TK, Scher
HI, Rathkopf DE, Halabi S, Armstrong AJ, Beebe DJ, Yu M,
Sundling KE, Taplin ME, Lang JM: A clinical-grade liquidbiomarker detects neuroendocrine differentiation in prostate cancer.
J Clin Invest 132(21): e161858, 2022. DOI: 10.1172/JCI161858
6 Antonarakis ES, Lu C, Wang H, Luber B, Nakazawa M, Roeser JC,
Chen Y, Mohammad TA, Chen Y, Fedor HL, Lotan TL, Zheng Q,
De Marzo AM, Isaacs JT, Isaacs WB, Nadal R, Paller CJ, Denmeade
SR, Carducci MA, Eisenberger MA, Luo J: AR-V7 and resistanceto enzalutamide and abiraterone in prostate cancer. N Engl J Med
371(11): 1028-1038, 2014. DOI: 10.1056/NEJMoa1315815
7 Duijvesz D, Luider T, Bangma CH, Jenster G: Exosomes asbiomarker treasure chests for prostate cancer. Eur Urol 59(5):823-831, 2011. DOI: 10.1016/j.eururo.2010.12.031
8 Ji J, Chen R, Zhao L, Xu Y, Cao Z, Xu H, Chen X, Shi X, Zhu
Y, Lyu J, Jiang J, Wang Y, Zhou T, He J, Wei X, Wu JB, Yang
B, Wang F: Circulating exosomal mRNA profiling identifies
novel signatures for the detection of prostate cancer. Mol Cancer
20(1): 58, 2021. DOI: 10.1186/s12943-021-01349-z
9 Del Re M, Biasco E, Crucitta S, Derosa L, Rofi E, Orlandini C,
Miccoli M, Galli L, Falcone A, Jenster GW, Van Schaik RH,
Danesi R: The detection of androgen receptor splice variant 7 inplasma-derived exosomal RNA strongly predicts resistance tohormonal therapy in metastatic prostate cancer patients. Eur Urol71(4): 680-687, 2017. DOI: 10.1016/j.eururo.2016.08.012

CANCER GENOMICS & PROTEOMICS 20: 456-468 (2023)

25 Nacarkahya G, Borazan E, Horozoglu C, Yaylim I: Investigationof long non-coding RNAs H19 and LINC00675 in colorectal
cancers in terms of histopathological features and correlations
with plasma markers. Anticancer Res 42(3): 1301-1306, 2022.
DOI: 10.21873/anticanres.15597
26 Rolla M, Jawiarczyk-PrzybyŁowska A, KolaČkov K,
Bolanowski M: H19 in endocrine system tumours. Anticancer
Res 41(2): 557-565, 2021. DOI: 10.21873/anticanres.14808
27 Singh N, Ramnarine VR, Song JH, Pandey R, Padi SKR, Nouri
M, Olive V, Kobelev M, Okumura K, McCarthy D, Hanna MM,
Mukherjee P, Sun B, Lee BR, Parker JB, Chakravarti D, Warfel
NA, Zhou M, Bearss JJ, Gibb EA, Alshalalfa M, Karnes RJ,
Small EJ, Aggarwal R, Feng F, Wang Y, Buttyan R, Zoubeidi A,
Rubin M, Gleave M, Slack FJ, Davicioni E, Beltran H, Collins
C, Kraft AS: The long noncoding RNA H19 regulates tumor
plasticity in neuroendocrine prostate cancer. Nat Commun 12(1):7349, 2021. DOI: 10.1038/s41467-021-26901-9
28 Xie Y, Ning S, Hu J: Molecular mechanisms of neuroendocrine
differentiation in prostate cancer progression. J Cancer Res Clin
Oncol 148(7): 1813-1823, 2022. DOI: 10.1007/s00432-02204061-7
29 Beltran H, Prandi D, Mosquera JM, Benelli M, Puca L, Cyrta J,
Marotz C, Giannopoulou E, Chakravarthi BV, Varambally S,
Tomlins SA, Nanus DM, Tagawa ST, Van Allen EM, Elemento O,
Sboner A, Garraway LA, Rubin MA, Demichelis F: Divergent
clonal evolution of castration-resistant neuroendocrine prostatecancer. Nat Med 22(3): 298-305, 2016. DOI: 10.1038/nm.4045
30 Asberry AM, Liu S, Nam HS, Deng X, Wan J, Hu CD:
Reprogramming landscape highlighted by dynamic transcriptomesin therapy-induced neuroendocrine differentiation. Comput Struct
Biotechnol J 20: 5873-5885, 2022. DOI: 10.1016/j.csbj.2022.10.031
31 Wang J, Yang K, Yuan W, Gao Z: Determination of serum
exosomal H19 as a noninvasive biomarker for bladder cancer
diagnosis and prognosis. Med Sci Monit 24: 9307-9316, 2018.
DOI: 10.12659/MSM.912018
32 Zhou H, Shen W, Zou H, Lv Q, Shao P: Circulating exosomal
long non-coding RNA H19 as a potential novel diagnostic andprognostic biomarker for gastric cancer. J Int Med Res 48(7):300060520934297, 2020. DOI: 10.1177/0300060520934297
33 Zhong G, Wang K, Li J, Xiao S, Wei W, Liu J: Determinationof serum exosomal H19 as a noninvasive biomarker for breast
cancer diagnosis. Onco Targets Ther 13: 2563-2571, 2020. DOI:
10.2147/OTT.S243601
34 Wako K, Kawasaki T, Yamana K, Suzuki K, Jiang S, Umezu H,
Nishiyama T, Takahashi K, Hamakubo T, Kodama T, Naito M:
Expression of androgen receptor through androgen-converting
enzymes is associated with biological aggressiveness in prostatecancer. J Clin Pathol 61(4): 448-454, 2008. DOI: 10.1136/
jcp.2007.050906
35 Necchi A, Cucchiara V, Grivas P, Bratslavsky G, Jacob J, Spiess
PE, Sokol ES, Killian JK, Lin D, Ramkissoon S, Huang RSP,
Madison RW, Venstrom JM, Schrock AB, Danziger N, Decker
B, Gjoerup O, Graf RP, Oxnard GR, Tukachinsky H, Ross JS:
Contrasting genomic profiles from metastatic sites, primarytumors, and liquid biopsies of advanced prostate cancer. Cancer
127(24): 4557-4564, 2021. DOI: 10.1002/cncr.33865
36 Traish AM, Morgentaler A: Epidermal growth factor receptor
expression escapes androgen regulation in prostate cancer: apotential molecular switch for tumour growth. Br J Cancer
101(12): 1949-1956, 2009. DOI: 10.1038/sj.bjc.6605376

10 Pandey GK, Kanduri C: Long non-coding RNAs: Tools forunderstanding and targeting cancer pathways. Cancers (Basel)
14(19): 4760, 2022. DOI: 10.3390/cancers14194760
11 An C, Wang I, Li X, Xia R, Deng F: Long non-coding RNA in
prostate cancer. Am J Clin Exp Urol 10(3): 170-179, 2022.
12 Li Y, Ji J, Lyu J, Jin X, He X, Mo S, Xu H, He J, Cao Z, Chen X,
Xu Y, Wang L, Wang F: A novel urine exosomal lncRNA assay to
improve the detection of prostate cancer at initial biopsy: aretrospective multicenter diagnostic feasibility study. Cancers(Basel) 13(16): 4075, 2021. DOI: 10.3390/cancers13164075
13 Işın M, Uysaler E, Özgür E, Köseoğlu H, Şanlı Ö, Yücel ÖB,
Gezer U, Dalay N: Exosomal lncRNA-p21 levels may help todistinguish prostate cancer from benign disease. Front Genet 6:168, 2015. DOI: 10.3389/fgene.2015.00168
14 Motamedinia P, Scott AN, Bate KL, Sadeghi N, Salazar G, Shapiro
E, Ahn J, Lipsky M, Lin J, Hruby GW, Badani KK, Petrylak DP,
Benson MC, Donovan MJ, Comper WD, McKiernan JM, Russo
LM: Urine exosomes for non-invasive assessment of gene
expression and mutations of prostate cancer. PLoS One 11(5):e0154507, 2016. DOI: 10.1371/journal.pone.0154507
15 Wang Y, Ji J, Wang B, Chen H, Yang Z, Wang K, Luo C, Zhang
W, Wang F, Zhang X: Tumor-derived exosomal long noncoding
RNAs as promising diagnostic biomarkers for prostate cancer. Cell
Physiol Biochem 46(2): 532-545, 2018. DOI: 10.1159/000488620
16 Kawakami K, Fujita Y, Kato T, Horie K, Koie T, Umezawa K,
Tsumoto H, Miura Y, Katagiri Y, Miyazaki T, Ohsawa I,
Mizutani K, Ito M: Diagnostic potential of serum extracellular
vesicles expressing prostate-specific membrane antigen in
urologic malignancies. Sci Rep 11(1): 15000, 2021. DOI:
10.1038/s41598-021-94603-9
17 Hishida S, Kawakami K, Fujita Y, Kato T, Takai M, Iinuma K,
Nakane K, Tsuchiya T, Koie T, Miura Y, Ito M, Mizutani K:
Proteomic analysis of extracellular vesicles identified PI3K pathway
as a potential therapeutic target for cabazitaxel-resistant prostatecancer. Prostate 81(9): 592-602, 2021. DOI: 10.1002/pros.24138
18 Bostick M, Bolduc N, Lehman A, Farmer A: Strand-specifictranscriptome sequencing using smart technology. Curr Protoc
Mol Biol 116(1), 2016. DOI: 10.1002/cpmb.22
19 Cancer Genome Atlas Research Network: The moleculartaxonomy of primary prostate cancer. Cell 163(4): 1011-1025,
2015. DOI: 10.1016/j.cell.2015.10.025
20 Tummala R, Nadiminty N, Lou W, Evans CP, Gao AC: Lin28
induces resistance to anti-androgens via promotion of AR splicevariant generation. Prostate 76(5): 445-455, 2016. DOI: 10.1002/
pros.23134
21 Puca L, Vlachostergios PJ, Beltran H: Neuroendocrine
differentiation in prostate cancer: emerging biology, models, andtherapies. Cold Spring Harb Perspect Med 9(2): a030593, 2019.
DOI: 10.1101/cshperspect.a030593
22 Zhu ML, Kyprianou N: Androgen receptor and growth factor
signaling cross-talk in prostate cancer cells. Endocr Relat Cancer
15(4): 841-849, 2008. DOI: 10.1677/ERC-08-0084
23 Brannan CI, Dees EC, Ingram RS, Tilghman SM: The productof the H19 gene may function as an RNA. Mol Cell Biol 10(1):28-36, 1990. DOI: 10.1128/mcb.10.1.28-36.1990
24 Wu B, Zhang Y, Yu Y, Zhong C, Lang Q, Liang Z, Lv C, Xu F,
Tian Y: Long noncoding RNA H19: a novel therapeutic target
emerging in oncology via regulating oncogenic signaling
pathways. Front Cell Dev Biol 9: 796740, 2021. DOI:
10.3389/fcell.2021.796740

467

Kato et al: EV-H19 as a Maker for ARAT-resistance
41 Kang HY, Lin HK, Hu YC, Yeh S, Huang KE, Chang C: Fromtransforming growth factor-beta signaling to androgen action:
identification of Smad3 as an androgen receptor coregulator inprostate cancer cells. Proc Natl Acad Sci USA 98(6): 3018-3023,
2001. DOI: 10.1073/pnas.061305498
42 Shiota M, Fujimoto N, Matsumoto T, Tsukahara S, Nagakawa S,
Ueda S, Ushijima M, Kashiwagi E, Takeuchi A, Inokuchi J, Uchiumi
T, Eto M: Differential impact of TGFB1 variation by metastatic
status in androgen-deprivation therapy for prostate cancer. Front
Oncol 11: 697955, 2021. DOI: 10.3389/fonc.2021.697955

37 Li P, Chen J, Miyamoto H: Androgen receptor signaling in
bladder cancer. Cancers (Basel) 9(2): 20, 2017. DOI: 10.3390/
cancers9020020
38 Lin X, Shi Q, Yang XJ: Cytomorphology, immunoprofile, andclinicopathologic correlation of metastatic prostatic carcinoma. Hum
Pathol 130: 36-46, 2022. DOI: 10.1016/j.humpath.2022.10.007
39 Sommer U, Siciliano T, Ebersbach C, Beier AK, Stope MB,
Jöhrens K, Baretton GB, Borkowetz A, Thomas C, Erb HHH:
Impact of androgen receptor activity on prostate-specificmembrane antigen expression in prostate cancer cells. Int J Mol
Sci 23(3): 1046, 2022. DOI: 10.3390/ijms23031046
40 Kaewput C, Vinjamuri S: Update of PSMA theranostics inprostate cancer: current applications and future trends. J Clin
Med 11(10): 2738, 2022. DOI: 10.3390/jcm11102738

Received May 22, 2023
Revised June 28, 2023
Accepted July 3, 2023

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