# DDX5 mRNA-targeting antisense oligonucleotide as a new promising therapeutic in combating castration-resistant prostate cancer

Thi Khanh Le, Chaïma Cherif, Kenneth Omabe, Clément Paris, François Lannes, Stéphane Audebert, Emilie Baudelet, Mourad Hamimed, Dominique Barbolosi, Pascal Finetti, et al.

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## To cite this version:

Thi Khanh Le, Chaïma Cherif, Kenneth Omabe, Clément Paris, François Lannes, et al. DDX5 mRNA-targeting antisense oligonucleotide as a new promising therapeutic in combating castration-resistant prostate cancer. Molecular Therapy, 2023, 31 (2), pp.471-486. 10.1016/j.ymthe.2022.08.005. hal-04391851

HAL Id: hal-04391851

https://inria.hal.science/hal-04391851v1

Submitted on 31 Mar 2025

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Distributed under a Creative Commons Attribution 4.0 International License
DDX5 mRNA-targeting Antisense Oligonucleotide as a new promising therapeutic in
combating Castration-Resistant Prostate Cancer

1 Thi Khanh Le ¹,², Chaïma Cherif ¹, Kenneth Omabe ¹, Clément Paris ¹, Francois Lannes ¹,

2 Stéphane Audebert ³, Emilie Baudelet ³, Mourad Hamimed ⁴, Dominique Barbolosi ⁴, Pascal

3 Finetti ¹, Cyril Bastide ⁵, Ladan Fazli ⁶, Martin Gleave ⁶, François Bertucci ¹, David Taïeb ¹, ⁷
4 and Palma Rocchi ¹, ⁸*

5

6 ¹Predictive Oncology Laboratory, Centre de Recherche en Cancérologie de Marseille, Inserm

7 UMR 1068, CNRS UMR 7258, Institut Paoli-Calmettes, Aix-Marseille University, 27 Bd. Leï

8 Roure, 13273 Marseille, France

9 ²Department of Life Science, University of Science and Technology of Hanoi, Hanoi 000084,
10 Vietnam

10

11 ³Marseille Protéomique, Centre de Recherche en Cancérologie de Marseille, INSERM,
12 CNRS, Institut Paoli-Calmettes, Aix-Marseille University, 13009 Marseille, France

12

13 ⁴ Inria – Inserm team COMPO, COMPUTational pharmacology and clinical Oncology, Centre
14 Inria Sophia Antipolis – Méditerranée, Centre de Recherches en Cancérologie de Marseille,
15 Inserm U1068, CNRS UMR7258, Institut Paoli-Calmettes, Aix-Marseille University, 27
16 Boulevard Jean Moulin - 13005 Marseille, France

15

16

17 ⁵ Urology Department, AM-HP Hospital Nord, Aix-Marseille University, 13915 Marseille
cedex 20, France

18

19 ⁶The Vancouver Prostate Centre, University of British Columbia, Vancouver, BC V6H 3Z6
20 Canada

20

21 ⁷La Timone University Hospital, Aix-Marseille University, 13005 Marseille, France

22 ⁸European Center for Research in Medical Imaging, Aix-Marseille University, 13005
23 Marseille, France

22

23
24 * Correspondence should be addressed to P.R (palma.rocchi@inserm.fr)

25 Tel: +33 4 86 97 72 67; Fax +33 486 977 499; Email: palma.rocchi@inserm.fr

26 Running title: DDX5-targeting ASO as a promising therapeutic for CRPC treatment
Abstract

The Heat Shock Protein 27 (Hsp27) has emerged as a principal factor of the Castration-
Resistant Prostate Cancer (CRPC) progression, also, an Antisense Oligonucleotide (ASO)
against Hsp27 (OGX-427 or Apatorsen) has been assessed in different clinical trials. Here,
we illustrate that Hsp27 highly regulates the expression of the human DEAD-box protein 5
(DDX5), and define DDX5 as a novel therapeutic target for CRPC treatment. DDX5
overexpression is strongly correlated with aggressive tumor features, and notably with CRPC.
DDX5 downregulation using a specific ASO-based inhibitor which acts on DDX5 mRNAs
inhibits cell proliferation in pre-clinical models, and particularly restores the treatment
sensitivity of CRPC. Interestingly, through the identification and analysis of DDX5 protein
interaction networks, we have identified some specific functions of DDX5 in CRPC that
could contribute actively to tumor progression and therapeutic resistance. We first present the
interactions of DDX5 and the Ku70/80 heterodimer and the transcription factor IIH (TFIIH),
thereby uncovering DDX5 roles in different DNA repair pathways. Collectively, our study
highlights critical functions of DDX5 contributing to CRPC progression and provides
preclinical proof-of-concept that a combination of ASO-directed DDX5 inhibition with a
DNA damage-inducing therapy can serve as a highly potential novel strategy to treat CRPC.

Keywords

Antisense Oligonucleotides (ASOs); Castration-Resistant Prostate Cancer (CRPC); DDX5;
DNA Damage Response (DDR); Protein Interactions.
## Introduction

Prostate Cancer (PC) is the second most frequent cancer and the fifth common cause of cancer deaths in men¹. Most patients with advanced PC are treated with standard Androgen Deprivation Therapy (ADT). Despite the initial response to ADT, patients tend to suffer from disease evolution to a castration-resistant state (CRPC) with pejorative outcomes². The heat-shock protein 27 (Hsp27) was found upregulated in the vast majority of CRPC cases and mediates tumor growth and drug resistance. Hsp27 inhibition by Antisense Oligonucleotide (ASO) and small interference RNA (siRNA) have revealed to significantly restore tumor sensitivity to castration and chemotherapy in preclinical models³⁻⁵. We previously illustrated that Hsp27 stabilizes its critical oncogenic interacting-proteins such as Eukaryotic translation Initiation Factor 4E (eIF4E), Translationally Controlled Tumour Protein (TCTP) and menin through its chaperon activites⁶⁻⁸. To get insight into the network of proteins or cellular functions that are regulated by Hsp27, a large-scale proteomic analysis was performed in our laboratory and suggested that DDX5 is an Hsp27-mediated protein in PC⁸. In addition, the established Hsp27 interactions identified DDX5 as a potential novel Hsp27-interacting protein in PC cells but not in the non malignant prostatic cells (L.T.K, unpublished data). DDX5 acts as an oncoprotein in a wide range of cancers, and links to drug response in many studies⁹,¹⁰. Together, these observations lead us to hypothesize that Hsp27 could confer CRPC emergence through DDX5, thus DDX5 could serve as a high potential therapeutic target for CRPC treatment.

DDX5, a member of the DEAD-box RNA helicase family, plays central roles in gene expression *via* participating in cellular RNA metabolism such as mRNA decay, ribosome biogenesis, splicing and transcription ¹¹,¹². DDX5 functions as a transcriptional co-activator for a number of highly regulated transcription factors, including estrogen receptor α (ERα) ¹³, β-catenin ¹⁴, androgen receptor (AR) ¹⁵, p53 ¹⁶. DDX5 is over-expressed in nearly 93% of
human cancers¹⁰ such as breast cancer ¹⁷ and prostate cancer ¹⁸. In addition, DDX5 has been
demonstrated to enhance cell proliferation, metastasis, and resistance to drugs through
stimulating different oncogenic signaling mechanisms ⁹,¹⁰.

In PC, DDX5 acts as an AR-co activator of transcription ¹⁵. DDX5 interacts with β-
Catenin, a transcriptional co-regulator of AR, which increases the AR transcriptional activity
remarkably ¹⁵. DDX5 was shown to promote cell survival and growth in AR-negative PC cell
lines (DU-145, PC-3) by promoting mTORC1 signaling ¹⁹. The oncogenic lncRNA CCAT1
was demonstrated to activate AR-mediated genes through interacting with DDX5, thereby
promoting PC cell proliferation ²⁰. The previous study reported that DDX5 overexpression is
linked to PC ¹⁸; however, whether or not DDX5 expression level is up-regulated in CRPC, it
remains undefined.

This work aims to further characterize the oncogenic roles of DDX5 in PC, providing
perspectives of ASO-directed DDX5 silencing in CRPC treatment. We show that Hsp27
positively regulates DDX5 protein levels, which in turn are associated with tumor growth and
progression. The Tissue Microarray (TMA) analysis implies that DDX5 is a novel therapeutic
target for CRPC remedy due to a strong positive correlation between DDX5 overexpression
and CRPC. An ASO selectively targeting DDX5 mRNAs (DDX5-ASO, WO2021099394A1,
2021) is developed and illustrated in its high efficiency in the downregulation of DDX5
expression and cell proliferation both in vitro and in vivo. In addition, we determine and
analyse DDX5 protein interactions using four human prostate cell lines (PNT1A, LNCaP,
DU-145 and PC-3) recapitulating different disease stages. Compared to the normal prostate
cell (NM) and castration sensitive (CS) cells, the DDX5 interaction proteins in the castration
resistant (CR) cells are exclusively enriched in functions mainly related to DNA damage
response, transcription regulation, RNA stability, and DNA conformation changes, which
could promote tumor progression and therapeutic resistance in PC. Interestingly, we identify a
95 new critical role of DDX5 in DNA damage repair in CRPC, and demonstrate that DDX5

96 inhibition via DDX5-ASO enhances treatment sensitivity, which opens new therapeutic

97 perspectives for PC patients.
RESULTS

Hsp27 highly regulates DDX5 expression via preventing DDX5 protein from proteasomal degradation

Our recent works based on proteomic approaches have suggested that DDX5 is a Hsp27-regulated protein 8 and probably interacts with Hsp27 in PC cells (L.T.K, unpublished data). Here, we confirmed that the DDX5 protein level is higher in the LNCaP-Hsp27 than in LNCaP-mock cells (Figure 1A). Hsp27 depletion by OGX-427 results in remarkable decreases of DDX5 levels in both LNCaP and PC-3 cell lines (Figure 1B). However, DDX5 mRNA expression is not altered under both conditions of Hsp27 upregulation and downregulation (Figure 1C), implying that Hsp27 does not regulate DDX5 expression either at transcriptional level or through controlling DDX5 mRNAs stability.

On the other hand, the interaction between Hsp27 and DDX5 is verified by WB following IP in PC-3 cells (Figure 1D). Since the ubiquitin-proteasome system was previously reported to mediate DDX5 degradation 21,22, we examined whether DDX5 protein stability is regulated by the 26S proteasome in PC. The CR PC-3 cells were treated with either MG132 or Bortezomib (proteasome inhibitors) with or without cycloheximide (CHX, an inhibitor of de novo protein synthesis). DDX5 levels increased in the presence of the proteasome inhibitors (Figure 1E), meanwhile they were stable by the combination of both MG132 and CHX (Figure 1F). These results indicate that DDX5 degradation is mainly modulated by the proteasomal machinery in PC. Furthermore, adding Bortezomib reverses the downregulation effect of OGX-427 on DDX5 protein levels (Figure 1G), suggesting that binding of Hsp27 to DDX5 can protect DDX5 protein from degradation by the proteasome.

To evaluate whether both proteins have mutual effects, we evaluated the Hsp27 protein expression upon DDX5 silencing. Hsp27 protein level was not changed due to DDX5
inhibition (Figure 1H). Therefore, Hsp27 controls DDX5 expression and DDX5 does not regulate Hsp27 expression.

**Elevated DDX5 protein expression is strongly associated with PC progression and castration-resistant phenotype**

We examined DDX5 expression by immunohistochemistry (IHC) in a large tissue microarray series of different stages of PC specimens. As shown in **Figure 2A&B**, PC has a significantly higher level of DDX5 protein expression than benign prostatic hyperplasia (BPH). Although we found DDX5 upregulation in high Gleason scores, no statistical significance was observed, certainly due to tumor heterogeneity and the small number of samples in the Gleason 5 group (n=32). Among the 266 PC specimens analyzed, Gleason grade 5 PC has the strongest DDX5 expression, followed by Gleason grade 4 and 3 (Figure 2C, D). Of note, DDX5 expression is remarkably upregulated in CRPC, with an average of DDX5 staining intensity is 10.56 times and 7.3 times higher than in Hormone naïve and Neoadjuvant Hormonal Therapy (NHT) tumors, respectively (Figure 2E, F). This is in an agreement with DDX5 overexpression observed in CR cell lines (DU-145 and PC-3) compared with the CS LNCaP cell line (Figure 2G). The DDX5 subcellular localization was persistent across all the specimens. Furthermore, high expression levels of DDX5 mRNA positively correlated with poor recurrence-free survival (RFS), with 10 year-RFS of group "DDX5 high" and "DDX5 low" are 34 % and 63%, respectively (p= 1.94E-02, Figure 2H). Taken together, these results indicate that DDX5 could represent a novel potential target for CRPC treatment.

**ASO-directed DDX5 silencing significantly decreases CRPC proliferation in both in vitro and in vivo models**
146

To specifically inhibit DDX5 expression, we first designed 93 ASOs potentially acting on the human DDX5 mRNA sequence (hASO) (Excel S1 & Figure S1). The 1st screening testing 13 hASOs showed that hASO51 inhibits effectively DDX5 expression (Figure 3A). Subsequently, the screening of DDX5-ASOs was continued to find out a bi-specific sequence of both human and murine forms (hmASOs), allowing us to identify potential toxic effects of DDX5 inhibition in xenografted mice. We tested 8 bi-specific sequences distributed uniformly over the entire target sequence at different locations of the mRNA in addition to hASO51. The hmASO3 is shown to downregulate effectively DDX5 protein expression (Figure 3B) and also in dose-dependent manner as hASO51 (Figure 3C). Moreover, DDX5 inhibition induced by both hASO51 and hmASO3 results in significant decreases of cell viability in CR PC-3 (Figure 3D), and in two LNCaP-derived cell lines (Figure S2).

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Subsequently, we assess the effect of DDX5 downregulation via DDX5-ASO (hmASO3) on the development of PC-3 xenograft tumors. The mice group treated with DDX5-ASO have a significant delay in tumor growth compared to the group treated with Control-ASO (***p<0.001, using repeated measures ANOVA) (Figure 3E, Table S1& Figure S3). Furthermore, the population pharmacodynamic modeling approach was used to characterize the tumor growth kinetics and to quantify the effect of the ASO treatment on tumor size evolution ²³. The Gompertz model was selected as it can reflect the sigmoidal nature of growth, and fit to the observed data significantly better than other models (Figure 3F, left & middle panels & Supplemental Methods). The inclusion of the treatment groups in the model produced a statistically significant drop in the objective function (ΔOFV = 11, p-value for likelihood ratio test < 0.001), demonstrating that tumor growth was inhibited significantly in the DDX5-ASO treatment group compared to the control group. Model predictions showed that individuals for whom the ASO treatment was administered, showed a 20% decrease in maximum tumor volume (Figure 3F, right panel). During the experiment, the mice were
healthy (SI, Excel S3) and did not appear to be on the reverse side-effects regarding animal behaviours (data not shown).

The identified DDX5 protein interactions reveal a broad functional landscape of DDX5 in PC

To decipher how DDX5 could promote PC progression, we determined and analyzed protein interaction networks of the endogenous DDX5 in different human PC cells using Immunoaffinity purification mass spectrometry (IP-MS). In total, 489 DDX5-associated proteins were identified, and we obtained 16, 44, 239, and 401 interaction candidates for PNT1A, LNCaP, DU-145, and PC-3, respectively (with the selection filter FDR 0.01, So = 1, SI, Excel S2). Obviously, a correlation of the number of DDX5 binding-proteins with the disease aggressiveness and CRPC was observed, suggesting that DDX5 may acquire more functions during PC progression. Of note, there are 48 candidates (12%) characterized as known DDX5 interactors in the published database (String.db), including very well-documented DDX5-interacting proteins such as TP53 ¹⁶, DHX9 ²⁴, CDK9 ²⁵ (Figure 4A). In addition, our identified interactome of DDX5 reinforces the interaction between DDX5 and Hsp27.

Classifying the DDX5-binding proteins based on the PANTHER 14.1 "Molecular function" and "protein class" category shows that a majority of the DDX5 interactors are involved in binding to nucleic acid and most of them are RNA-binding proteins (Figure S4). It also reveals several protein classes engaging with catalytic functions and illustrates a set of DDX5-associated proteins which act as transcription factors. According to Gene Ontology Biological Process (GO-BP) enrichment analysis, 126 and 79 biological functions are enriched with the significant level p = 0.05 and 0.005, respectively (Figure S5 & Excel S2). As expected, a number of well-documented functions of DDX5 are observed (Figure 4B).
Accordingly, we found that DDX5 plays a vital role in gene expression since a majority of DDX5 interactors (up to 57%) are engaged with this process (p = 0.0000E-100). DDX5 functions mainly in RNA processing (p = 1.2751E-72), translation (p = 1.8605E-71), ribosome biogenesis (p = 1.8027E-42) and transcription (p = 2.8988E-6). In addition, DDX5 modulates gene expression at posttranscriptional levels by regulating mRNA stability, RNA splicing, and translation. DDX5 is also found to participate in DNA topological change (p = 1.2599E-8), and the cellular response to the DNA damage stimulus (p = 4.4587E-3) including DNA repair (p = 4.4272E-3).

By performing a functional enrichment analysis using Gprofiler with the CORUM 3.0 database for the identified DDX5-associated proteins, various enriched protein complexes corresponding to different functions are determined (Figure 4C). In agreement with GO-BP enrichment analysis, DDX5 was found to be tightly associated with the established protein complexes involved in ribosome biogenesis, protein synthesis in both cytoplasm and mitochondria, splicing, mRNA stability, transcription, and DNA repair (Table S2). In particular, DDX5 was found to be tightly associated with the IGF2BP2/IGF2BP3 complex (mRNA stability), Toposome (cell cycle), the general transcription factor complex GTFIIH (transcription initiation & DNA repair), and 7SK RPN complex (transcription elongation) (Figure 4D).

### Acquired biological functions of DDX5 in CR cells contribute to the switch from castration-sensitive PC to castration-resistant PC

Comparison of the enriched GOs obtained from DDX5 PPI networks in 4 cell lines using the ClusterProfiler R packages gives us clues about DDX5 functions promoting tumor progression and CRPC. Obviously, DDX5 was involved in much more biological functions in CR cells than in both CS and NM (Figure S6). Only three GO terms are common for both CS
and CR cells, that are related to mRNA alternative splicing and mitochondrial translation.
This implies that DDX5 could play a role in these two biological functions in both CSPC and
CRPC. DDX5 could drive tumor progression and treatment resistance through its implications
in DNA damage response, mRNA stabilization, transcription and DNA conformation changes
since these are the main cellular processes exclusively enriched in CRPC cells compared to
CSPC (Figure 5A).

To verify several key interactions identified by MS analysis, IPs followed by western blot
analysis are carried out. Accordingly, DDX5 is confirmed to interact with the Ku70/Ku86
heterodimer (DNA repair, Figure 5B), NF45 (pri-ribosomal RNA processing, DNA repair,
Figure 5B), GTF2H1 (transcription, DNA repair, Figure 5C), and YBX1 (mRNA stability,
Figure 5D).

232
233 DDX5 inhibiton with ASO technology restores treatment sensitivity in CR cells

A set of DDX5-binding proteins participating in DNA damage response are determined
and most of them are exclusively found in the CRPC cells and implicated in two DNA repair
pathways: Non-homologous End Joining (NHEJ) and Nucleotide excision repair (NER)
(Table 1). To decipher the role of DDX5 in the DNA repair, we examine the effect of DDX5
silencing on DNA repair recovery following radiations. The irradiation induced foci (IRIF)
numbers (i.e., double-strand breaks) are higher in the DDX5-depleted cells compared to the
control samples (DDX5 proficient cells) at 1hr, 7hr, and 24hr following irradiations (Figure
6A, B). In parallel with IF, WB analysis using both total and subcellular protein extracts
illustrated that DDX5 knockdown decreases DNA repair efficiency (Figure 6C). Moreover,
DDX5-ASO significantly enhances sensitivity to either irradiation or cisplatin compared to
the control-ASO in CRPC DU-145 cells (Figure 6D, E). Particularly, combining DDX5-ASO
with cisplatin can remarkably lower the GI50 of cisplatin up to nearly 3 times compared with
246 the combination of Control-ASO and cisplatin (3.838 vs 9.601 µM) (Figure 6E). All together

247 imply that an overexpressed DDX5 could enhance DNA damage response and genome

248 maintenance in CRPC, promoting tumor progression and treatment resistance (Figure 6F).
Discussion

Nucleic acid (NA)-based technologies are currently developing rapidly with more than 249 15 NA drugs approved by the FDA and EMA and more than 2000 NA therapies undergoing 250 clinical investigations 26,27. Compared with siRNAs, ASO has several advantages such as its 251 lower cost of production and easier synthesis and cell penetration without any transfecting 252 agents 28. Due to remarkable successes of mRNA-based vaccine during the current COVID- 253 19 pandemic, mRNA targeting will represent a promising growing field in the next few years 254 for personalized therapy in oncology. DDX5 has been identified as a cellular target of RX- 255 5902 29 and Resveratrol 19. However, clinical translations of these agents have been hindered 256 by certain limitations including lack of specificity. RX-5902 has not been tested in PC so far 257 and probably does not work with AR-negative PC because the β-catenin/DDX5 interactions 258 are AR-dependent in PC 15. As with many plant-based phytochemicals, the biggest challenge 259 of resveratrol in clinical trials is its poor bioavailability once administered 30. For these 260 reasons, the DDX5 mRNA-directed ASO can serve as the most effective DDX5-targeting 261 therapy for PC patients. 262

The obtained interaction maps of DDX5 in CR cells showed that DDX5 interacts with a 263 number of critical oncogenic proteins linked to tumor progression, and drug resistance in 264 various malignancies such as IGF2BPs 31, YBX1 32,33, YY1 34,35 and CTCF 36. Most notably, 265 we uncovered novel DDX5-interacting proteins involved in different DNA repair pathways in 266 PC. DDX5 has been previously reported to engage in DNA damage response 16. This was 267 attributed via recruitment of both p53 and RNAPII to the p21 promoter upon irradiation 268 stress, resulting in cell cycle arrest after DNA damage. DU-145 cells harbor p53 mutation 269 which was believed to produce non-functional protein meanwhile the PC-3 are null-TP53 cell 270 line 37; therefore, DDX5 might respond to the irradiation stimulates in a p53-independent 271 manner in the PC-3 and DU-145 cells. 272
273 Emerging evidences highlight the central roles of DDX5 and other the RNA-interacting
274 proteins in genome stability, notably in DNA repair ³⁸,³⁹. R-loops or DNA-RNA hybrids are
275 generated not only at active transcription sites by RNA polymerase but also around DNA
276 break sites, and play a fundamental function in DNA repair ⁴⁰. Aberrant R-loop formation can
277 result in replication stress, DNA double-strand breaks and genomic instability ⁴¹,⁴². DDX5
278 associates with DNA:RNA hybrids ⁴³, unwinds unscheduled R-loops ⁴², and DDX5
279 downregulation accumulates R-loop genome-wide and particularly at transcription start sites
280 (TSSs), transcription termination sites in chromosome regions with very high gene density ⁴⁴
281 and at DSBs ⁴⁵. Furthermore, DDX5 has been previously illustrated to promote DNA damage
282 repair through acting as an active player in resolving R-loops near or at DSBs, thereby
283 maintaining genomic stability ⁴¹,⁴⁵. These findings imply the functions of DDX5 in
284 transcription (both transcription initiation and termination), DNA repair and genome
285 maintenance via R-loops regulation.

286 On the other hand, the present work suggests the dual functions of DDX5 in transcription
287 and DNA damage response that can be achieved through the DDX5 interaction with the
288 multifunctional TFIIH complex and the Ku DNA end-binding proteins. There are many lines
289 of scientific evidence supporting a cross-talk between transcription and DNA repair via a set
290 of common molecular utilized-network such as the TFIIH core subunits XPD and XPB ⁴⁶,⁴⁷,
291 and the Ku70/80 complex that has been recently reported to bind to the promoter DNA region
292 and interact with transcription factors for transcriptional modulation ⁴⁸,⁴⁹.

293 Emma L. Clark et al. pointed out that DDX5 functions as a “coupling” factor,
294 coordinating both transcription and RNA processing of AR-regulated genes in PC ¹⁵. In
295 consistency with this finding, our DDX5 PPI revealed the tight connection between DDX5
296 and the core proteins of the basal initiation transcription factor complex TFIIH playing a role
297 in transcription of protein-coding genes mediated by RNA Pol II ⁴⁷. Several protein subunits
of the transcription factor IIIC complex and the RNAP (RNA polymerase) were obtained in the DDX5 interactome of CR cells. Noticeably, our data also showed a group of transcription factors which appeared as DDX5 interacting proteins (data not shown). In the absence of AR - negative CR cells, cellular survival and growth are driven by by-pass pathways, including increased activity of MAPK, PI3K, PCK cascade, and aberrant expression and activity of oncogenes (such as BCL2). DDX5 could participate in expression regulation of these oncogenes by coordination with other transcription factors, transcriptional machinery and with RNA-processing proteins, thereby contributing to CRPC development.

Collectively, the oncogenic functions of DDX5 in PC that were described in the present work and previous reports allow us to paint a hypothesis picture of how DDX5 can promote CRPC by playing as a cross-talk among different signaling pathways such as transcription, RNA processing, and DNA repair. Accessible chromatin regions of highly transcribed genes located in gene-rich areas frequently have increased R-loop formation<sup>44,50</sup> and more fragile to DNA damage inducing-stimuli than the heterochromatin part of the genome<sup>51</sup>. As a result, it is crucial for the CR cells to resolve R-loop structures and to response promptly and properly to DNA damages occurring in the genomic regions with higher transcription intensity to ensure genome maintenance. Without any therapeutics, DDX5 regulates R-loop structure, recruits and/or associates with RNAPII, transcription initiation complex (TFIIH) and other transcription factors, and participates in coupling transcription-RNA process, thereby facilitating transcription process, avoiding DNA damages, and maintaining genome intergrity. On the other hand, upon to DNA lesions inducing-treatments, DDX5 can immediately facilitate DNA repair at the active transcription sites either *via* clearing of RNA transcripts of R-loop or enhancing the recruitment and/or activity of the DNA repair machinery (Ku complex, TFIIH complex), ensuring proper and prompt DNA repair, thereby maintaining genomic stability and enhancing tolerance of cancer cells to therapies (Figure 7).
In summary, treatment options of CRPC that progress after chemotherapy remains challenging. Here, we show the translational significance of DDX5 silencing using ASO technology that could be represented as a new therapeutic strategy in CRPC. DDX5 overexpression is linked to CRPC progression and DDX5 can promote disease evolution through participating in various critical cellular processes, notably in the DNA damage response. Our worldwide-patented ASO targeting DDX5 could have potential to be highly impactful in combating CRPC, particularly in the setting of CRPC progressing after Docetaxel treatment, as a monotherapy or more probably in combination with other approaches including other chemotherapies or therapeutic radiation (e.g, PSMA-directed radionuclide therapy). Additionally, this ASO- directed DDX5 mRNA silencing could be operative in other cancer types in which DDX5 acts as a major oncogenic player. Further works will focus on evaluating toxicologic and pharmacologic properties of the DDX5 ASO.
## Cell lines and cell culture conditions

Four different human prostate cell lines were used as a way to study PC pathogenesis :
normal phenotype cell PNT1A (NM, ECACC, European Collection of Cell Cultures,
England), castration-sensitive (CS) LNCaP (ATCC, American Type Culture Collection
(Rockville, MD, USA), and castration-resistant (CR) DU-145 and PC-3 cell lines (ATCC).
Additionally, two LNCaP-derived cell lines were used, LNCaP overexpressing Hsp27
(LNCaP-Hsp27) and LNCaP-mock (empty vector) which were established as previously
described ⁷. The cells were maintained with either the RPMI-1640 medium (Roswell Park
Memorial Institute) (for PNT1A, LNCaP, LNCaP-mock and LNCaP-Hsp27) or DMEM
medium (Dulbecco's Modified Eagle's Medium) (for PC-3 and DU-145), supplemented 10%
fetal bovine serum (FBS) at 37 °C in 5% CO2.

## Human prostate cancer TMA construction and immunohistochemistry (IHC)

A TMA (tissue microarray) containing 515 specimens [79 benign prostate hyperplasia, 266 untreated primary (hormone naïve) tumors, 113 neoadjuvant hormone therapy tumors, 57 castration-resistant prostate cancers (CRPC)] was obtained from the Vancouver Prostate Centre Tissue Bank. Except CRPC samples that were obtained *via* transurethral resection of the prostate (TURP), all specimens were collected through radical prostatectomy. IHC staining was performed by Dr. Ladan Fazli with mouse anti-DDX5 monoclonal antibody (sc-365164, Santa Cruz Biotechnology, CA, USA) using the Ventana autostainer (model Discover XT; Ventana Medical System, Tucson, AZ) with an enzyme-labelled biotin-streptavidin system and a solvent-resistant DAB Map kit (Ventana). DDX5 staining intensity was scored using Aperio/Leica software called ImageScope. The image analyzing process was done by applying IHC (intensity and pixel count) scoring algorithms directly to the annotated area in the scanned image.
ASO design, selection and ASO transfection in PC cells

Synthesis of DDX5-targeting ASOs was performed in the “Optoligo”, a French Inserm Transfert-sponsored platform. ASOs were designed using a computational algorithm developed in our laboratory by P. Finetti. Firstly, the coding part of the DDX5 mRNA sequence (NM_001320595.2) was selected and divided into a set of consecutive sequences that contained 20 base pairs. The resulting sequences were converted into their complementary acid nucleotide sequences, which were subsequently inverted to obtain the potential ASO sequences. The ASO sequences were individually evaluated regarding their GC content and their specificity using NCBI's 'Basic Local Alignment Search Tool' (BLAST), with the use of the 'blastn' algorithm and the NCBI reference transcript database 'refseq_rna' as parameters. The ultimate selection was done manually by excluding all ASOs showing sequence similarities with other genes. The lead ASOs were synthesized in our laboratory by C. Paris following a previously described procedure <sup>52</sup>. These ASOs has been modified with phosphorothioate (PS) backbone that protect them against degradation by nucleases. The ASOs alter targeted mRNA through RNase H1-mediated RNA cleavage.

Cells (at ~50% density) were transfected with ASOs two times as previously described <sup>52</sup>. ASOs were pre-incubated with 1 mL Gibco Opti-MEM (Life Technologies SAS, Courtaboeuf, France) containing 4 uL Oligofectamin (Life Technologies SAS, Courtaboeuf, France) for 20-30 minutes before adding to the cells. After 4 hr, the mixture of ASOs and Oligofectamin was removed and the fresh completed medium was supplied. The ASO treatment was repeated exactly the same on the following day. After 3 days, the transfected cells were harvested for protein extraction and Western blot analysis (WB). The protein expression was evaluated quantitatively using Image J (NIH) software.
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The hmASO3 was patented worldwide by Inserm Transfer with the PATENT N.REF as PCT/EP2020/082548 (WO2021099394A1, 2021).

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## Preclinical evaluation of ASO treatment in PC-3 xenografts

387

PC-3 cells ($10^6$) in 100 µl DBPS 1X were inoculated subcutaneously in the right flank of 5-week-old male BALB/c Nude mice (Charles River Laboratories, France). After 12 days, the tumors reached from 50 to 100 mm³ and the treatment started on the next day. Mice (n=14) were randomly selected for treatment with DDX5- or control-ASO. Each group received daily intraperitoneal (IP) injection of 12.5 mg/kg DDX5- or control-ASO for 5 weeks. Tumor volume (mm³) was measured weekly using a calliper in three perpendicular dimensions (x = width, y = length, z = depth), and calculated following the formula $x * y * z * 0.5236$. During the period of treatment, the mice were observed for signs of systemic toxicity and their body weights were daily recorded too. The mice were maintained in animal facility (agreement #13.2700). Dr Palma Rocchi owns a personal agreement (#A13-477) for the animal handling and experimentation for this study. Animal experimentation was performed following to ethics laws recommendations.

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400

## Western blot analysis (WB)

401

WB experiments were carried out following the described procedure⁵². The membranes were incubated with the primary antibodies including Vinculin and GAPDH as loading controls (SI, Table S1). The primary Abs were probed with the corresponding HRP-conjugated secondary Abs (DAKO, Agilent, United States) and detected using ECL Prime Western Blotting detection reagent (RPN2236, GE Healthcare, Vélizy-Villacoublay, France). The images were captured using the Amersham Imager 680 blot and gel imager (GE Healthcare, Vélizy-Villacoublay, France).

402

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405

406

407
408 Immunofluorescence for DNA damage foci formation

409 The DU-145 cells were cultured on coverslips. After 3 days of transfection with the
410 DDX5 ASO as described above, the cells were irradiated using RS-2000X-RayIrradiator (Rab
411 Source Technology Inc., USA). Subsequently, the cells were fixed in 4% (wt/vol)
412 paraformaldehyde (PAF) for 15 min, permeabilized with 0.1% Triton X-100 in 1X PBS for 15
413 min, blocked with 5% (wt/vol) bovine serum albumin (BSA) for 30 min, at room temperature
414 (RT). The samples were then incubated overnight at 4°C with primary antibody against p-
415 γH2AX, followed by 1h-incubation at RT with a secondary FITC anti-rabbit antibody (Alexa
416 Fluor 488, Thermo Fisher Scientific, Life Technologies SAS, Villebon-sur-Yvette, France).
417 Nuclei were stained with DAPI (4', 6-diamidino-2-phenylindole) and coverslips were
418 mounted with ProLong™ Gold Antifade Mountant (Thermo Fisher Scientific, Life
419 Technologies SAS, Villebon-sur-Yvette, France). Images were captured using Apotome
420 (Zeiss, Le Pecq, France).

421

422 Bioinformatics analyses

423 The DDX5-interacting proteins found in 4 cell lines were functional classified using
424 PANTHER 14.1 <sup>53</sup>. Analysis of Gene Ontology (GO) enrichment using BiNGO tool run by
425 Cytoscape <sup>54</sup> with the statistical test "Hypergeometric test", and Benjamini & Hochberg False
426 Discovery Rate (FDR) correction, two significant levels p = 0.05 and p = 0.005, and used the
427 whole annotation as a reference set. Analysis of Protein complexes enrichment was performed
428 using Gprofiler <sup>55</sup> for CRORUM 3.0 database (<a href="https://mips.helmholtz-muenchen.de/corum/">https://mips.helmholtz-muenchen.de/corum/</a>)
429 which collects annotation of mammalian protein complexes obtained from manual
430 experiments <sup>56</sup> with threshold p=0.05. To compare the GO enrichment analysis results among
431 four DDX5 interactomes from four cell lines, the ClusterProfiler R packages <sup>57</sup> were used.
432 DDX5 mRNA expression was extracted from TCGA PRAD RNAseq public data
(https://portal.gdc.cancer.gov/projects/TCGA-PRAD). *DDX5* in 498 tumour samples (T) was measured as discrete value after comparison with median expression in 52 normal prostate samples (N): upregulation, thereafter designated “*DDX5* high” was defined by a T/N ratio ≥1 and no upregulation (“*DDX5* low”) by a T/N ratio <1.

## Statistical analysis

All pooled results are represented as mean ± SEM. Statistical analyses were carried out using GraphPad Prism 6 software (GraphPad Software, Inc). The independent sample t test was performed to compare means between two groups. Recurrence-free survival (RFS) from TCGA PRAD samples was calculated from the date of diagnosis until the date of relapse. Follow-up was measured from the date of diagnosis to the date of last news for event-free patients. Results with *P* < 0.05 were considered statistically significant and are indicated by *, **, P < 0.01; ***, P < 0.001, and ****, P < 0.0001.
## Funding

445 This work was supported by Inserm and grants from ITMO Cancer (BioSys call, #A12171AS) and the Amidex Fondation 'Emergence & Innovation' project (NucleoTHER).

446 K.L.T fellowship was funded by the 911 project-Vietnam International Education Department (VIED). Proteomic analyses were done using the mass spectrometry facility of Marseille

448 Proteomics (marseille-proteomique.univ-amu.fr) supported by IBISA (Infrastructures Biologie Santé et Agronomie).

451
452 Acknowlegement

453 We would like to give special thanks to the Experimental Histo-Pathology platform (IPC/CRCM Experimental Pathology or ICEP) for the help with Ki67 immunohistological

455 staining, to Dr. Pauline BRIGE (European Center for Research in Medical Imaging, Aix-Marseille University) for her help with the Ki67 staining analysis, and to Mr. DUONG Quang

457 Hieu (M2 student, Inserm UMR 1068, CNRS UMR 7258, Institut Paoli-Calmettes, Aix-Marseille University) for his support with Western blot analysis during the revision.

459 Author contributions
460 T.K.L., D.T. and P.R. conceived this project; T.K.L., C.C., K.O., C.P., F.R., S.A., E.B., L.F. performed and analyzed the experiments; T.K.L., M.H., D.B., P.F. and C.B. performed bioinformatics analyses; D.T., P.R., M.G., F.B. gave critical comments; T.K.L.wrote the manuscript with the approval of all other authors.

Declaration of Interests

464 Authors declare to have no financial, personal or professional competing interest, and no conflict of interest

465
466
Data Availability :
467 Protein interaction AP-MS data: PRIDE PXD023252

468 (http://www.ebi.ac.uk/pride/archive/projects/PXD023252).
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<table><caption>Table 1. List of DDX5-interaction proteins implicated in the DNA damage response identified in 4 PC cell lines</caption><thead><tr><th>Proteins</th><th>PNT1A</th><th>LNCaP</th><th>DU-145</th><th>PC-3</th><th>DDR_related Pathways</th></tr></thead><tbody><tr><td>XRCC6</td><td></td><td></td><td>x</td><td>x</td><td>NHEJ</td></tr><tr><td>XRCC5</td><td></td><td></td><td>x</td><td>x</td><td>NHEJ</td></tr><tr><td>GTF2H4</td><td></td><td></td><td>x</td><td>x</td><td>NER</td></tr><tr><td>GTF2H1</td><td></td><td></td><td>x</td><td>x</td><td>NER</td></tr><tr><td>ERCC3</td><td></td><td></td><td>x</td><td>x</td><td>NER</td></tr><tr><td>GTF2H2</td><td></td><td></td><td>x</td><td>x</td><td>NER</td></tr><tr><td>GTF2H3</td><td></td><td></td><td>x</td><td>x</td><td>NER</td></tr><tr><td>RFC5</td><td></td><td>x</td><td>x</td><td>x</td><td>NER</td></tr><tr><td>UPF1</td><td></td><td></td><td>x</td><td>x</td><td>unclear</td></tr><tr><td>PRPF19</td><td></td><td></td><td>x</td><td>x</td><td>NER, NHEJ</td></tr><tr><td>IGHMBP2</td><td></td><td></td><td>x</td><td>x</td><td>unclear</td></tr><tr><td>MSH6</td><td></td><td>x</td><td>x</td><td></td><td>MR</td></tr><tr><td>TP53</td><td></td><td></td><td>x</td><td></td><td>BER, NER, DSBR, mitochondrial DNA repair</td></tr><tr><td>LIG3</td><td></td><td></td><td>x</td><td></td><td>BER, NER, alNHEJ, HR, mitochondrial DNA repair</td></tr><tr><td>RFC3</td><td></td><td></td><td>x</td><td></td><td>NER</td></tr></tbody></table>
<table><tr><td>ERCC2</td><td></td><td>x</td><td></td><td>NER</td></tr><tr><td>ERCC6</td><td></td><td></td><td>x</td><td>NER</td></tr><tr><td>RFC1</td><td></td><td></td><td>x</td><td>NER</td></tr><tr><td>TOP2A</td><td></td><td></td><td>x</td><td>DDR</td></tr><tr><td>RECQL4</td><td></td><td></td><td>x</td><td>HR</td></tr><tr><td>MRPS26</td><td></td><td></td><td>x</td><td>mitochondrial DNA repair</td></tr><tr><td>MRPS35</td><td></td><td></td><td>x</td><td>mitochondrial DNA repair</td></tr><tr><td>DTX3L</td><td></td><td></td><td>x</td><td>NHEJ</td></tr></table>

640

641 Footnote: DSBR: double strand break repair, NHEJ: Non homologous end-joining, HR: Homologous

642 Recombination, MR: Mismatch Repair, BER: Base excision repair, NER: Nucleotide Excision

643 Repair, TC – NER: Transcription coupled Nucleotide Excision interacting proteins of DDX5 play a

644 role in DNA repair according to GO repair
Figure legends

<caption>Figure 1. Hsp27 positively regulates DDX5 expression through preventing DDX5 protein from proteasomal degradation</caption>

A, Western blotting (WB) analyzing the protein expression levels of DDX5, Hsp27 in LNCaP-mock versus LNCaP-Hsp27 (overexpressed Hsp27) shows that DDX5 expression is higher in LNCaP-Hsp27 compared to the LNCaP-mock. B, WB analyzing the protein expressions of DDX5 in the LNCaP and PC-3 cells transfected with OGX-427 (an Hsp27-targeting ASO) or Control-ASO reveals that DDX5 protein expression decreased dramatically upon Hsp27 depletion. C, Hsp27 does not regulate DDX5 at transcriptional level. RT-qPCR analysis showed that DDX5 mRNA levels are similar in both LNCaP-mock and LNCaP-Hsp27 cells. The mRNA levels of both DDX5 and Hsp27 are evaluated by RT-qPCR analysis using the PC-3 cells transfected with OGX-427 or Control-ASO. The DDX5 mRNA level is constants upon Hsp27 knock down. Sample t test was performed to compare the relative mRNA levels between two samples (LNCaP-mock versus LNCaP-Hsp27, OGX-427 versus control-ASO-transfected PC-3 cells. **, p < 0.01; ns, non-significant. D, Confirmation of the interaction between Hsp27 and DDX5. DDX5 IP followed by WB analysis showed that Hsp27 was present in the IP using anti-DDX5 antibody. E, DDX5 stability is controlled by the proteasome pathway in PC. Proteasome inhibition using either 10 µM MG132 or 100 nM Bortezomib induces accumulation of DDX5 protein at the indicated times. F, Proteasome inhibitor combined with an inhibitor of de novo protein synthesis, CHX (Cycloheximide) conserves DDX5 protein levels. G, Hsp27 prevents DDX5 from proteasome degradation. PC-3 cells were transfected with OGX-427 or Control-ASO at 150 nM concentration for 2 days, followed by incubation with or without 100 nM Bortezomib for 24 hours. WB analysis shows that the proteasome inhibitor Bortezomib can reverse the downregulation of DDX5 induced by OGX-427. H, DDX5 does not control Hsp27 protein abundance. WB analyses the DDX5
669 and Hsp27 expression upon either Hsp27 knock down by OGX-427 or DDX5 downregulation
670 by DDX5-ASO in PC-3 cells. Hsp27 protein expression is stable upon DDX5 depletion.

671 **Figure 2. DDX5 overexpression is associated with tumor progression and CRPC as**
672 **clinically relevant**

673 **A**, DDX5 expression is significantly higher in prostate cancer (PC) compared to benign
674 prostatic hyperplasia (BPH). **B**, representative images of DDX5 staining show increased
675 DDX5 expression in PC specimens compared to BPH. **C**, results show a tendency for DDX5
676 expression to increase with increasing Gleason grade. **D**, representative images of DDX5
677 staining show increased staining intensity in Gleason grade 5 specimens. **E**, the graph and the
678 representative IHC images show that DDX5 expression is extremely higher in castration-
679 resistant prostate cancers (CRPC) patients than in the hormone naïve and neoadjuvant
680 hormone therapy (NHT) tumors. **F**, representative images of DDX5 staining show an
681 remarkable increase of DDX5 staining expression in CRPC specimens. **G**, DDX5 is
682 overexpressed in CR cells (DU-145 & PC-3) compared to CS cells (LNCaP). The DDX5
683 protein level was analyzed by Western blot using total cell lysate from 3 different prostate cell
684 lines (LNCaP, DU-145 and PC-3). DDX5 protein level was normalized with vinculin which
685 was also used as loading control. Quantification illustrates that DDX5 expression levels in
686 both CR lines are at about 3 times as high as in CS cells. **H**, Kaplan-Meier RFS curves in the
687 TCGA PRAD public samples. High expression level of DDX5 mRNAs positively correlated
688 with poor recurrence-free survival (RFS). According to the DDX5 mRNA levels, samples
689 were classified into two groups, DDX5 high and DDX5 low, that have 10 year-RFS rates of
690 34 % and 63%, respectively in a pool of 490 informed patients.

691 *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, non significant.

692
Figure 3. ASO-directed DDX5 downregulation inhibits cell proliferation and PC-3 xenograft-tumor growth

A, B, C, ASO screenings aim to define ASOs that inhibit DDX5 protein expression. Both hASO51 (human ASO) and hmASO3 (human/mouse) decrease DDX5 expression in dose-dependent manner. D, DDX5-knock down by both hASO51 and hmASO3 significantly inhibit cell proliferation. The MTT test was done with biological triplication, and the data was analyzed using an independent sample t-test. ***, P < 0.001. E, Mean (±SE) growth curve of PC-3 xenografted tumors (***p<0.001, using repeated measures ANOVA). Male BALB/c Nude mice harboring PC-3-derived xenografted tumor (7 mice/group) were daily I.P (intraperitoneal) injected with 12.5 mg/kg Control-ASO or DDX5-ASO (hmASO3) for 5 weeks. Tumor volume (mm³) was measured weekly using a calliper in three perpendicular dimensions (x = width, y = length, z = depth), and calculated following the formula x * y * z * 0,5236. The group treated with DDX5-ASO have a delay in tumor growth compared to the group of Control-ASO. F, Population pharmacodynamic modeling demonstrates that DDX5-ASO treatment inhibits significantly the tumor growth compared to the control group. Two above panels presents the Goodness-of-fit and predictive check (VPC) plot. Overall, these two plots indicate that the base model provided an acceptable fit to the observed data. **Left panel**: Individual predicted volumes versus observed volumes. Points represent individual predicted volumes plotted against observed volumes and are distributed evenly around the identity line, indicating that no major bias was observed across predicted values. **Middle panel**: Results of the scatter visual predictive check from 500 model-generated datasets. Solid triangles represent the observed volumes and are almost within the 95% prediction interval of the model, demonstrating that the model is able to reproduce the pharmacodynamic (PD) observations adequately. **Right panel**: Model prediction of tumor growth kinetics, depending
717 on the received treatment, using the final model. Individuals for the mice having treated with
718 DDX5-ASO showed a 20% decrease in maximum tumor volume.

719 SCR : scrambled oligonucleotide ; NT : Non - treated ; TSmax, individual carrying capacity
720 (predicted maximum tumor volume).

721
722 **Figure 4. Functional bioinformatics analyzing DDX5 interaction proteins identified in**
723 **the study**

724 A, The pie chart shows the proportion of the novel DDX5-interacting proteins (87.9%) and
725 the identified interactions characterized as known DDX5-interacting proteins in the string
726 database (12.1%). B, Gene Ontology-Biological Process (GO-BP) enrichment analyses of the
727 DDX5 interactome were performed using BiNGO (Hypergeometric test, Benjamini &
728 Hochberg False Discovery Rate (FDR) correction, p = 0.005, Biological Process). The bar
729 chart shows the top functions ranked by the fold enrichment score. C, CORUM enrichment
730 analysis allows identifying experimental-proved human proteins complexes associated with
731 DDX5. D, Network modeling of the top enriched protein complexes and DDX5. Green nodes
732 represent proteins found in the DDX5 interactome obtained in the study while grey nodes
733 mean proteins were not identified. Bordered edges represent interactions with DDX5
734 annotated in published PPI databases. All of the protein members of the IGF2BP1 complex
735 were found in the DDX5 interactions. A solid connection between DDX5 and the Toposome
736 was observed since most of the protein components of toposome associate with DDX5 in the
737 study (SSRP1 was not appeared in the DDX5 interactions but it was annotated to interact with
738 DDX5 in string db). We first showed the rigid association of DDX5 with the general
739 transcription factor complex GTFIIH (or TFIIH) which is composed of the core complex
740 (GTF2H1, GTF2H2, GTF2H3, GTF2H4, ERCC2, ERCC3) and the CAK (CDK7, CCNH,
741 MNAT1). DDX5 could modulate transcription is through the 7SK RPN complex because all
742 of its constructive proteins (CDK9, HEXIM1, CCNT1, and LARP7) interact with DDX5.

743 **Figure 5. Acquired DDX5 functions lead switching from Castration sensitivity to**
744 **Castration resistance of PC**

745 **A**, The scheme describing the hypothesis about how DDX5 can promotes PC progression and
746 therapeutic resistance basing on functional analyses of DDX5-interaction network. DDX5
747 could drive PC progression and therapeutic resistance through playing roles in DNA damage
748 response, mRNA stabilization, transcription, DNA conformation changes since these cellular
749 processes are exclusively enriched in the DDX5 PPI obtained in CRCP. In particular, DDX5
750 regulates mRNA stability *via* interacting with IGF2BPs complex, thereby modulating
751 expression of target genes. DDX5 also regulates gene expression by monitoring transcription
752 initiation *via* associating with the basal transcription complex TFIIH and transcription factors
753 (TFs). DDX5 functions in non-homologous end joining (NHEJ) *via* interacting with the
754 Ku70/Ku80 and NF45/NF90 complexes. DDX5 also interacts with the TFIIH complex and
755 RFC proteins (RFC1, RFC3, RFC5) to participate in Nucleotide Excision Repair (NER).
756 DDX5 may regulate cell cycle through its interaction with the Toposome complex acting in
757 chromosome segregation. Through binding to different topoisomerase enzymes such as
758 TOP2A, TOP2B and TOP1, DDX5 can modulate the DNA topological changes, thereby
759 monitoring DNA replication, transcription. **B**, IP using anti-DDX5 Ab followed by WB with
760 anti-Ku70 Ab, anti-Ku80 Ab, anti-NF45 Ab shows the presence of Ku70, Ku80 and NF45 in
761 the DDX5 complexes in both CR DU-145 and PC-3 cells (left above panel). The reverse IP
762 using anti-Ku70 Ab and anti- Ku80 Ab followed by WB using anti-DDX5 Ab confirmed the
763 interaction of DDX5 with the Ku70/Ku80 heterodimer (right above and below panel). **C, D,**
764 Similarly, the IP using anti-GTF2H2 Ab and anti-YBX1 Ab combined with WB using anti-
765 DDX5 Ab confirmed the association of DDX5 with GTF2H2 (C) and YBX1 (D).
766 **Figure 6.** DDX5 promotes therapeutic resistance of CRPC through activation of DNA repair
767

768 **A & B**, DDX5 downregulation reduces DNA damage repair efficiency by Immunofluorescent (IF) and irradiation induced foci (IRIF) counting. The DU-145 cells were transfected with 150 nM DDX5-ASO (hmASO3) or Control-ASO, and three days later, the cells were exposed to 5 Gy IR and further cultured for various time periods (UIR = Unirradiated, 1hr, 7hr, 24hr). IF staining using anti-γH2AX antibody was carried (A). The graph shows a higher number of the IRIF in DDX5-ASO-treated cells compared to the Control-ASO-transfected cells (B). **C**, **WB** analysis shows that DDX5 depletion defects the DNA repair process. DU-145 cells transfected with the DDX5-ASO (hmASO3) or Control-ASO (150 nM) were exposed to 5 Gy IR and further cultured for 7 hr. The total protein extract or compartment extracts including cytoplasmic and nuclear extracts was subjected to WB analysis using anti-DDX5 Ab, anti-γH2AX Ab and GAPDH used as a loading control. **D**, **E**, DDX5 knock-down enhances cell sensitivity to DNA damage stress stimuli such as irradiation (D) and cisplatin (E). The DU-145 cells were transfected with DDX5-ASO for 2 days, and treated with either irradiation or cisplatin, and subjected to cell proliferation evaluation using MTT assay after 2 days. The MTT tests were performed with triplication. DDX5-ASO can lower GI₅₀ of cisplatin up to nearly 3 times. **F**, In DDX5-overexpressed PC cells, DDX5 facilitates two main DNA repair pathways (NHEJ, NER), ensuring the proper DNA repair upon DNA damage-inducing therapies such as irradiation and chemotherapy, thereby promoting cell survival, PC development and therapeutic resistance (left panel). Conversely, DDX5 downregulated PC cells are failed to repair DNA lesions; therefore, they are sensitive to treatments (right panel).

788 **Figure 7.** The scheme illustrating a hypothesis picture of how DDX5 can promote tumor progression and therapeutic resistance *via* playing as a cross-talk among different signaling pathways such as transcription, RNA processing, and DNA repair in CRPC.
789
790
791 The increased R-loop formation frequently occurs in accessible chromatin regions of highly
792 transcribed genes located in gene-rich areas which more sensitive to DNA damage inducing-
793 therapies than the other part of the genome. Without any treatments, DDX5 mediates R-loops
794 structure, recruits and/or binds with RNAPII, transcription initiation complex (TFIIH) and
795 other transcription factors (TFs), and participates in coupling transcription-RNA process,
796 thereby facilitating transcription process, preventing DNA damages, promoting tumor
797 development. On the other hand, upon to DNA damage-inducing therapies, DDX5 can
798 immediately facilitate DNA repair at the active transcription sites either *via* clearance of RNA
799 transcripts of R-loop or enhancing the recruitment and/or activity of the DNA repair
800 machinery (Ku complex, TFIIH complex), ensuring proper and rapid DNA repair, resulting in
801 genomic maintenance and tolerance enhancement of cancer cells to therapies.
802
<figure><img src="image_3.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Figure 4</figcaption></figure>
<caption>Figure 5</caption>

A

<figure><img src="image_7.png" /></figure>

B

<figure><img src="image_8.png" /></figure>

C

<figure><img src="image_9.png" /></figure>

D

<figure><img src="image_10.png" /></figure>
<caption>Figure 6</caption>

A

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

B

<figure><img src="image_13.png" /></figure>

C

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

D

<figure><img src="image_16.png" /></figure>

E

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>
<figure><img src="image_20.png" /><figcaption>Figure 7</figcaption></figure>

<figure><img src="image_21.png" /></figure>
Hsp27 stabilizes DDX5 from proteasome-mediated degradation

<figure><img src="image_22.png" /></figure>

ASO-directed DDX5 mRNA knockdown inhibits cell proliferation and tumor growth

<figure><img src="image_23.png" /></figure>

DDX5 promotes PC progression and therapeutic resistance through activation of DNA damage response

Normal prostate cell (NM)

Castration-sensitive PC cell (CSPC)

Castration-resistant PC cell (CRPC)

<figure><img src="image_24.png" /></figure>

- Protein trimerization

<figure><img src="image_25.png" /></figure>

- Protein trimerization

<figure><img src="image_26.png" /></figure>

- Splicing

- Splicing

- Mitochondrial translation

- Mitochondrial translation

Main acquired DDX5 functions contributing tumor progression & treatment resistance in CRPC

- mRNA stability

- Transcription

- DNA conformation changes

- DNA damage response

DNA damage response

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>

Nucleotide Excision Repair (NER)

Genome maintenance

**Therapeutic resistance**
(chemotherapy, radiation therapy)

CRPC cells