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. 2023 Sep-Oct;20(5):456-468.
doi: 10.21873/cgp.20397.

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

Taku Kato  1   2 , Kyojiro Kawakami  3 , Kosuke Mizutani  4 , Tatsuya Ando  5 , Yasuhiro Sakai  5 , Kouhei Sakurai  5 , Shohei Toyota  2 , Hidetoshi Ehara  2 , Hiroyasu Ito  5 , Masafumi Ito  3
Affiliations

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

Taku Kato et al. Cancer Genomics Proteomics. 2023 Sep-Oct.

Abstract

Background/aim: We aimed to evaluate the changes of androgen receptor (AR) signaling-related long non-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 profiling of 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 role of H19, the prognosis was analyzed in PC patients and proteomic 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 score and up-regulated in neuroendocrine CRPC tissue with low AR expression. Furthermore, EV-H19 expression was significantly associated with worse outcome to androgen-deprivation therapy. Proteomic analysis demonstrated that H19 knockdown enhanced PC-related protein expression.

Conclusion: EV-H19 may negatively correlate with AR-signaling activity and could be a marker to diagnose ARAT-resistance among CRPC patients.

Keywords: CRPC; H19; Prostate cancer; RNA-sequencing; androgen receptor axis-targeted therapy; extracellular vesicles.

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Conflict of interest statement

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

Figures

Figure 1
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 descending order of the number of transcripts each transcription factor regulated.
Figure 2
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.
Figure 3
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.
Figure 4
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).
Figure 5
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) Proteins regulated 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.
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