Targeting CHD1L suppresses prostate cancer progression via the FOXO3-PUMA axis

Targeting CHD1L suppresses prostate cancer progression via the FOXO3-PUMA axis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Targeting CHD1L suppresses prostate cancer progression via the FOXO3-PUMA axis Pusheng Hui, Yanru Lai, Haiqi Fan, Qinrong Yan, Yue Yang, Zhe Chen, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7424447/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Dec, 2025 Read the published version in Journal of Translational Medicine → Version 1 posted 4 You are reading this latest preprint version Abstract Background Prostate cancer (PCa) is one of the most common malignancies in men worldwide, and advanced or metastatic disease remains a major therapeutic challenge. Chromodomain helicase DNA binding protein 1-like (CHD1L) has been implicated as an oncogenic driver in multiple cancer types, yet its role in prostate cancer pathogenesis is not fully defined. The purpose of this study is to investigate the biological significance of CHD1L in prostate cancer and to evaluate the therapeutic potential of its selective inhibitor OTI-611. Methods Bioinformatics analyses were conducted to assess the expression, prognostic significance of CHD1L in PCa patients. In vitro , cell viability, cycle progression, apoptosis, and migration/invasion were evaluated using CCK-8, colony formation, flow cytometry, transwell assays. In vivo treatment potential of OTI-611 was assessed through a nude mouse xenograft model. Protein and mRNA levels were determined by western blot and qPCR, respectively. Synergism of OTI-611 and docetaxel was determined using SynergyFinder 3.0. Results We demonstrated that CHD1L was significantly upregulated in PCa patients and correlates with poor prognosis. Genetic knockdown of CHD1L substantially inhibits PCa cell proliferation and induces apoptosis. Moreover, inhibition of CHD1L by the small molecule OTI-611 significantly suppresses PCa cell proliferation, migration, and invasion, and induces apoptosis both in vitro and in vivo . Mechanistically, inhibition of CHD1L induces the expression of FOXO3 (a classic transcription factor) and its downstream target PUMA (a key apoptosis inducer). Restricting the expression of FOXO3 significantly reverses the anti-tumor effects induced by OTI-611. Furthermore, OTI-611 synergizes with docetaxel to enhance apoptotic cell death, providing a promising strategy to overcome docetaxel resistance. Conclusions Our study demonstrates that CHD1L is markedly upregulated in prostate cancer and contributes to tumor progression. Pharmacological inhibition of CHD1L with the selective inhibitor OTI-611 significantly suppresses proliferation, migration, and invasion, while inducing apoptosis in vitro and in vivo . Mechanistically, these effects are mediated through activation of the FOXO3–PUMA axis, as FOXO3 suppression abrogates OTI-611–induced apoptosis. Moreover, OTI-611 exhibits strong synergy with docetaxel, enhancing apoptotic cell death and providing a potential strategy to improve therapeutic efficacy in prostate cancer. Prostate cancer CHD1L FOXO3 PUMA Docetaxel Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Prostate cancer (PCa) is the most common malignancy among men globally 1 . Although androgen deprivation therapy (ADT) is initially effective, most patients with advanced disease progress to castration-resistant prostate cancer (CRPC), where treatment options are limited and prognosis is poor 2 . Taxane-based chemotherapy, particularly docetaxel, improves survival in both metastatic hormone-sensitive and castration-resistant settings 3 , 4 . However, approximately 50% of patients initially respond to docetaxel, but nearly all develop resistance within 6–8 months of treatment 5 , highlighting the need for novel therapeutic strategies and effective combination regimens. Chromodomain helicase DNA-binding protein 1-like (CHD1L) is an ATP-dependent chromatin remodeler that regulates DNA repair, transcription, and cell survival pathways 6 , 7 . Aberrant CHD1L expression is implicated in several malignancies, where it promotes proliferation, metastasis, and therapy resistance 8 – 12 . Despite its well-established role in other cancers, the clinical significance and therapeutic potential of CHD1L in prostate cancer remain underexplored. OTI-611 is a small-molecule inhibitor of CHD1L that binds its ATPase/helicase domain, displaying potent antitumor activity through allosteric inhibition of CHD1L ATPase 13 , 14 . Although OTI-611 has demonstrated anti-tumor activity in other malignancies 15 – 17 , its efficacy and underlying mechanisms in prostate cancer remain to be fully elucidated. Forkhead Box O3 (FOXO3), a key tumor suppressor transcription factor, regulates apoptosis, cell cycle arrest, DNA damage repair, and stress responses 18 – 20 . Its pro-apoptotic target, PUMA (BBC3), is a potent BH3-only protein that triggers apoptosis 21 – 23 . Activation of the FOXO3-PUMA axis has been shown to induce robust apoptosis in prostate cancer cells 24 , 25 , making it an attractive therapeutic target. Here, we demonstrate that both knockdown and pharmacological inhibition of CHD1L using OTI-611 induce apoptosis in prostate cancer cells in vitro and inhibit tumor progression in vivo . Mechanistically, we identify the FOXO3-PUMA axis as a key mediator of OTI-611-induced apoptosis. Furthermore, we show that OTI-611 synergizes with docetaxel to enhance apoptotic cell death, providing a strong rationale for its use in combination therapy for advanced prostate cancer. Materials and methods Patient samples and cell culture All samples used in this study were acquired from 6 patients with PCa at The First Affiliated Hospital of Chongqing Medical University. This study was conducted under the guidelines of the Declaration of Helsinki and was approved by the Institutional Ethics Committee. The HEK293T cell line was obtained from the American TypeCulture Collection (ATCC). The prostate cancer cell lines DU145, PC3 ,22RV1 and LnCAP and the non-malignant prostate epithelial cell line RWPE1 were all obtained from Procell Life Science & Technology. Cell lines were reauthenticated through short tandem repeat analysis and assessed for mycoplasma contamination every six months following thawing in our experiments. DU145, PC3 ,22RV1 and LnCAP cells were cultured in MEM or RPMI 1640 medium supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Hyclone). The RWPE-1 cell line was cultured in K-SFM. HEK293T cells were maintained in DMEM containing 10% FBS and 1% penicillin/streptomycin. Chemical compounds For in vitro studies, OTI-611(CHD1Li 6.11, #HY-144256,MCE), and Docetaxel (#HY-B0011 ,MCE) were dissolved in DMSO to stock concentrations of 10mM. For in vivo experiments, OTI-611 was dissolved in 2% DMSO, 40%PEG300, 5% Tween-80, and 53% saline. Plasmids and lentivirus To generate the vectors for the expression of CHD1L-specific shRNA, we designed the sequence of shRNAs and cloned shRNAs into the vector pLKO.1-GFP-puro (refer to primer sequences in Supplementary Table S1 ). To generate the vectors for the expression of human CHD1L, the coding sequences were synthesized by Tsingke (China) and cloned into the vector pMSCV-IRES-EGFP. Small interfering RNAs (siRNAs) targeted FOXO3 were purchased from Tsingke (China) (refer to siRNA sequences in Supplementary Table S1 ). For virus production, HEK293T was transfected with lentiviral or retroviral plasmid with helper plasmid (psPAX2 and pMD.2G together with pLKO-shRNA vectors, pCL-Ampho together with pMSCV vectors). The medium was replaced with fresh medium at 12 h after transfection and culture supernatants were collected at 48 h and 72 h. The virus was stored at -80°C until use. Positively infected cells were isolated using flow cytometry sorting (GFP+) or puromycin treatment. Real-time quantitative reverse transcription PCR (RT-qPCR) The RT-qPCR was performed as described previously 55 . Generally, total RNA was isolated using the Total RNA Isolation Kit (Thermo Fisher) according to the manufacturer’s instructions. cDNA was reversetranscribed using PrimeScript RT reagent Kit (Takara) and subjected to real-time PCR with SYBR Green Supermix (Bio-Rad) in an iCycler iQ Real- Time PCR Detection System (Bio-Rad). All primers are listed in Supplementary Table S1 . All samples were run in triplicate. GAPDH was used as an internal control for mRNA. Cell proliferation and colony formation assays Cell proliferation was assessed using CCK8 kit (MA0218-5,Meilunbio) according to the manufacturer’s instructions. Cells (2× 10 3 ) suspended in 100 µl medium were seeded in triplicate in a 96-well plate, grown at 37°C. Medium containing 10% CCK8 replaced the original medium and incubated at 37°C for 2–4 h, and the absorbance was finally determined at 450 nm using a micro plate reader. For colony formation assay, 1 × 10 3 cells were cultured in 6-well plate at 37°C for 10–14 days, visible colonies were washed twice with phosphate-buffered saline (PBS), then fixed with 4% paraformaldehyde for 30 min following a suitable incubation period and subsequently stained with crystal violet. The number of colonies was counted visually. Flow cytometry analysis For the cell cycle analysis, 2 × 10 6 prostate cancer cells were washed with PBS and then fixed in 70% cold ethanol at 4°C overnight. Then, the cells were stained with DAPI (Biolegend, 422801) / RNase mixture in the dark at 37°C for 1 h. For the apoptosis assay, 1 × 10 6 prostate cancer cells were harvested, washed, and suspended in a binding buffer containing Annexin V-PE (Biolegend, 640947) and DAPI. All samples were analyzed by Flow cytometry on a BD FACS Canto II system, and all data were analyzed using FlowJo software. Western blot For the analysis of cell proteins, prostate cancer cells were lysed using RIPA lysis buffer (Beyotime). The resulting protein extracts were then separated by SDS-PAGE. The antibodies used in this study were against the following: rabbit anti-CHD1L(1:1000; #32923-1-AP; Proteintech), rabbit anti-Cyclin B1 (1:1000; #4138; CST), rabbit anti-Cyclin E1 (1:1000; #20808; CST), rabbit anti-Cdk1 (1:1000; #ET1607-51; HUABIO), rabbit anti-Cdk2 (1:1000; #ET1602-6; HUABIO), rabbit anti-BAX (1:1000; #2772; CST), rabbit anti-BCL-2 (1:1000; #3498; CST), rabbit anti-FOXO3 (1:1000; #2497; CST), rabbit anti-PUMA (1:1000; #98672; CST), rabbit anti-Tubulin (1:2000; #AF001; Beyotime). Secondary antibodies was HRP-conjugated Goat anti-rabbit IgG (H + L) (1:2500; #A0208;Beyotime). Cell migration and invasion assay Approximately 2 × 10 4 PCa cells were resuspended in 100 µL serum-free medium. Then the cells were inoculated into the upper transwell chamber of the insert (8-mm pores), and 600 µL of complete medium containing 20% FBS was added to the lower chamber. After 24h migration, the cells in low chambers were fixed with 4% paraformaldehyde for 30 min following a suitable incubation period and subsequently stained with crystal violet. Finally, random fields were imaged using an inverted microscope at 100× magnification. The number of cells in each image was calculated using the ImageJ software. Cell invasion studies were performed according to a similar Transwell method described above, except the upper chamber was pre-coated with Matrigel (356234,Corning). Transplantation of xenogeneic tumor cells Approximately 2× 10 6 PC3 cells were subcutaneously injected into the flanks of BALB/c nude mice (male, 8–10 weeks old) (day 0). After 10 days, mice were treated with vehicle or OTI-611 (20 mg/kg) every alternate day for a total of 8 days (days 10–18). Tumor volume was measured using vernier calipers every 2 days (days 10–20) and calculated with the formula: V = 0.5 × L × W 2 , where L is the longest diameter of the tumor and W is the longest transverse diameter perpendicular to L. On day 20, the mice were euthanized and the weights of the tumors were measured. The isolated tissues were immersed in 4% polyformaldehyde and sectioned at Aifang Biotechnology Co., Ltd. Tumor sections obtained from each group were stained with hematoxylin and eosin (H&E) (BP092; Biossci, Wuhan, China) to identify features of the xenograft tumors. Ki-67 (ab92742; Abcam, Cambridge, UK) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) (A112-03; Vazyme, Nanjing, China) were used to assess proliferation and apoptosis, respectively. RNA-sequencing and data analysis Total RNA samples were extracted from DU145 cells upon OTI-611 treatment. RNA-seq was performed by Sangon Science (Shanghai, China), and the libraries were sequenced by the Illumina HiSeq 2000 platform as 150-bp pair-ended reads. RNA-seq data were analyzed according to previous work 55 . Briefly, Fragments per kilobase per million (FPKM) estimation was performed with Cufflinks v2.1.1, aligned reads were counted with HTSeq (a Python framework to work with high-throughput sequencing data), and differential expression analysis was performed with DESeq2. Differentially expressed genes were identified based on a log2 fold change greater than 1 or less than − 1, accompanied by statistics. The volcano plot illustrating gene expression was generated using R. GO enrichment and pathway analyses was performed using the DAVID ( https://david.ncifcrf.gov/tools.jsp ) 56 and Metascape database ( http://metascape.org ) 57 . Gene set enrichment analysis was performed using GSEA software (v4.1.0) ( http://www.broadinstitute.org/gsea ) with 1000 permutations. Synergy score analysis The synergy score analysis was conducted using the web application SynergyFinder ( https://synergyfinder.fimm.fi ) 58 . The synergy score was determined by assessing the deviation from the theoretical inhibition calculated using the HSA method. According to the user guide available on the website, the synergy score is assessed as follows: scores less than − 10 indicate that the interaction between two drugs is likely to be antagonistic; scores ranging from − 10 to 10 suggest that the interaction is likely to be additive; and scores greater than 10 imply that the interaction is likely to be synergistic. Statistics analysis All experiments were repeated at least two times independently, and at least three independently samples were used for data analysis. We used simple randomization to allocate samples/animals to experimental groups. The sample size was not pre-selected, and no samples or animals were excluded from the analysis. Single-blind blinding was implemented in the experiment. The investigator was blinded to the group allocation during the outcome assessment. All statistical analysis was performed using GraphPad Prism 8 software (GraphPad Software). Statistical significance between the two groups was analyzed using a two-tailed unpaired Student’s t-test. For metaphase analysis, statistical comparisons were made using a one-way ANOVA test followed by Tukey–Kramer post-hoc tests. All data were expressed as mean ± standard deviation (SD). P values < 0.05 were considered statistically significant. Results CHD1L is upregulated in prostate cancer and correlates with poor prognosis To elucidate the potential role of CHD1L in prostate cancer (PRAD), we comprehensively assessed its expression across multiple publicly available datasets. In the TCGA-PRAD cohort, CHD1L was markedly upregulated in tumor tissues compared with normal prostate tissues (Fig. 1 A-B). These findings were consistently validated in independent GEO datasets, including GSE69545 and GSE72220 (Fig. 1 C-D). Analysis of the GSE2443 dataset revealed markedly higher CHD1L expression in AR-independent prostate cancer, an aggressive subtype with poorer prognosis 26 , compared with AR-dependent tumors (Figure. 1E), suggesting that CHD1L upregulation may contribute to androgen receptor-independent disease progression. Moreover, immunohistochemical (IHC) staining from the Protein Atlas database revealed moderate CHD1L staining in 16 of 20 PRAD samples, whereas all three normal samples displayed undetectable expression (Fig. 1 F). Clinically, elevated CHD1L expression was associated with significantly poorer disease-free survival (DFS), as demonstrated by Kaplan-Meier analysis (Fig. 1 G). Moreover, CHD1L expression was positively correlated with MKI67, a well-established proliferation marker (Fig. 1 H), implicating CHD1L in tumor growth progression. Consistently, increased CHD1L expression was observed at both mRNA and protein levels in prostate cancer cell lines (22RV1, PC3, and DU145) compared with the normal prostate epithelial cell line RWPE1 (Fig. 1 I-J). Paired clinical specimens further demonstrated significantly elevated CHD1L mRNA and protein levels in tumor tissues relative to adjacent normal tissues (Fig. 1 K-L). Collectively, these data demonstrate that CHD1L is frequently overexpressed in prostate cancer and may link to tumor progression. Knockdown of CHD1L suppresses proliferation, migration, invasion, and promotes apoptosis in prostate cancer cells To investigate the functional role of CHD1L in prostate cancer, we silenced CHD1L using two independent shRNAs in DU145 and PC3 cells. RT-qPCR and western blotting confirmed efficient CHD1L knockdown (Fig. 2 A-B). CCK-8 and colony formation assays demonstrated that CHD1L depletion markedly suppressed cell growth and clonogenic capacity (Fig. 2 C-F). Transwell assays further revealed that CHD1L knockdown significantly impaired both migration and invasion (Fig. 2 G-J). Moreover, Annexin V/DAPI flow cytometry showed increased apoptosis following CHD1L silencing (Fig. 2 K-L). Collectively, these data identify CHD1L as a critical oncogenic driver that promotes proliferation, migration, invasion, and survival of prostate cancer cells in vitro . OTI-611, a small molecule inhibitor of CHD1L, suppresses tumor cell growth and induces apoptosis in prostate cancer To assess the effects of pharmacologic CHD1L inhibition, we treated DU145 and PC3 cells with OTI-611 (CHD1Li 6.11), a small molecule inhibitor that binds to the ATPase/helicase domain of CHD1L 13 , 14 (Fig. 3 A). Dose-response analysis revealed that OTI-611 reduced cell viability in both cell lines, with IC 50 values of 4.380 µM and 3.963 µM, respectively (Fig. 3 B). 3 µM OTI-611 treatment significantly suppressed proliferation (Fig. 3 C-D) and decreased clonogenic capacity (Fig. 3 E-F). Transwell assays demonstrated that OTI-611 significantly impaired migration and invasion in both DU145 and PC3 cells (Fig. 3 G-J). Annexin V and DAPI staining revealed a robust increase in apoptotic cell populations (Fig. 3 K-L). It has been reported that OTI-611 impairs the cell-cycle progression in breast and colorectal cancer 16 , 17 . In this study, we observed that OTI-611 treatment led to an increased percentage of PCa cells in G2/M phase, associated with a reduced percentage of cells in G0/G1 phase (Fig. 3 M-N). Mechanistically, western blot analysis revealed that OTI-611 treatment resulted in changes of Cyclin B1, Cyclin E1, CDK1, and CDK2, along with increased expression of pro-apoptotic proteins BAX and reduced expression of the anti-apoptotic protein BCL2 (Fig. 3 O), further supporting the functional role of CHD1L in regulating cell cycle progression and apoptosis. To validate the specificity of OTI-611, we overexpressed CHD1L in PC3 cells (Fig. 3 P) and found that CHD1L overexpression partially rescued the OTI-611-induced growth suppression and apoptosis (Fig. 3 Q-S), confirming that the observed phenotypes are CHD1L-dependent. Together, these findings suggest that the pharmacologic inhibition of CHD1L by OTI-611 efficiently inhibits proliferation, migration and invasion and induces apoptosis of PCa cells in vitro . OTI-611 suppresses tumor growth in vivo without inducing systemic toxicity We next assessed the in vivo efficacy of OTI-611 using a PC3 xenograft model in BALB/c nude mice. Ten days after tumor implantation, mice were randomly assigned to receive intraperitoneal injections of OTI-611 (20 mg/kg) 11 or vehicle every other day for 8 days (Fig. 4 A). As the treatment progressed, OTI-611 markedly suppressed tumor growth, leading to significant reductions in tumor volume and weight compared with controls (Fig. 4 B-E). Histological examination further supported these findings. H&E staining of tumor sections revealed marked cell disintegration in the OTI-611 group (Fig. 4 F). In addition, Ki67 immunostaining showed a pronounced decrease in proliferative activity (Fig. 4 G), indicating reduced tumor cell growth in vivo . Consistent with our in vitro results, TUNEL staining demonstrated increased apoptotic cells in tumors from OTI-611-treated mice (Fig. 4 H). To evaluate safety, we performed histopathological analyses of major organs, including the heart, liver, spleen, lung, and kidney. No detectable abnormalities were observed (Fig. 4 I), suggesting that OTI-611 is well tolerated at the therapeutic dose. Taken together, these results confirm that pharmacologic CHD1L inhibition by OTI-611 effectively suppresses prostate cancer growth in vivo while exhibiting minimal systemic toxicity. OTI-611 activates FOXO3-mediated apoptotic signaling pathway in prostate cancer cells To investigate the molecular mechanisms underlying the anti-tumor effects of CHD1L inhibition, RNA sequencing was performed on DU145 cells treated with OTI-611 or vehicle, identifying 537 upregulated and 673 downregulated genes (Fig. 5 A). GO enrichment and pathway analyses indicated significant activation of transcriptional regulation, chromatin remodeling, DNA damage response, apoptotic signaling, and stress-related processes (Fig. 5 B-C). Among these, FOXO3 signaling was prominently enriched (Fig. 5 C). FOXO3 is a transcription factor regulating genes involved in apoptosis, proliferation, cell cycle progression, DNA damage repair, stress responses, and tumorigenesis 18 – 20 . In prostate cancer, FOXO3 functions as a tumor suppressor 18 , 27 – 29 , and its activation has been proposed as a therapeutic strategy 30 – 32 . PUMA (BBC3), a well-established FOXO3 transcriptional target 21 – 23 , was among the most highly upregulated genes after OTI-611 treatment. Previous studies have shown that activation of the FOXO3-PUMA axis induces potent apoptosis in prostate cancer cells 24 , 25 , underscoring its importance in therapeutic intervention. GSEA further demonstrated enrichment of TNFα signaling via NF-κB, p53 signaling, and unfolded protein response in OTI-611-treated cells (Fig. 5 D). Conversely, gene sets related to DNA repair, MYC targets, and interferon alpha response were negatively enriched, suggesting suppression of survival pathways. RT-qPCR confirmed that OTI-611 significantly increased FOXO3 and PUMA expression in DU145 and PC3 cells (Fig. 5 E). Western blotting validated a time- and dose-dependent upregulation of both proteins (Fig. 5 F), supporting activation of the FOXO3-mediated apoptotic pathway. Collectively, these findings identify the FOXO3-PUMA axis as a key downstream effector of CHD1L inhibition by OTI-611 in prostate cancer. FOXO3 knockdown attenuates the anti-tumor effects of OTI-611 in prostate cancer cells To determine whether FOXO3 is required for the anti-tumor activity of OTI-611, FOXO3 was silenced in DU145 and PC3 cells using three independent siRNAs, with siFOXO3-2 achieving the most efficient knockdown (Fig. 6 A-D). CCK-8 assays revealed that FOXO3 silencing largely rescued OTI-611-mediated growth inhibition (Fig. 6 E-F), while Annexin V/DAPI staining showed a marked reduction in OTI-611-induced apoptosis (Fig. 6 G-H). Western blotting demonstrated that FOXO3 knockdown significantly decreased PUMA expression, and that OTI-611-driven PUMA induction was abolished in FOXO3-deficient cells (Fig. 6 I-J). These findings identify FOXO3 as a pivotal mediator of OTI-611’s anti-cancer effects and highlight the FOXO3-PUMA axis as a central apoptotic pathway downstream of OTI-611 treatment in prostate cancer. OTI-611 synergizes with docetaxel to enhance apoptosis in prostate cancer cells Taxanes such as docetaxel are among the most active chemotherapies for advanced prostate cancer 5 , 33 , 34 , yet resistance inevitably develops in nearly all patients 5 . Given that CHD1L acts as a key anti-apoptotic factor in prostate cancer, we hypothesized that its inhibition by OTI-611 could enhance docetaxel efficacy. To test this, DU145 and PC3 cells were treated with escalating doses of docetaxel, OTI-611, or their combination. CCK-8 assays and heatmaps demonstrated that combination treatment produced greater growth inhibition than either agent alone (Fig. 7 A-B). SynergyFinder analysis confirmed a synergistic interaction (synergy score > 10) (Fig. 7 C-D). Annexin V/DAPI staining further showed that, while each drug alone induced moderate apoptosis, their combination markedly increased apoptotic cell populations in both cell lines (Fig. 7 E-H). These findings indicate that OTI-611 synergizes with docetaxel to promote apoptosis in prostate cancer cells, supporting its potential for combination therapy. Discussion Chromatin remodeling factors play a crucial role in tumorigenesis by regulating DNA repair, gene expression, and genomic stability, which are essential for tumor cell survival and proliferation under stress conditions 35 – 37 . CHD1L, an ATP-dependent chromatin remodeler 38 – 40 , has been implicated as an oncogene in multiple malignancies, including liver 41 , breast 42 , ovarian 9 , pancreas 43 , and lung cancer 44 . Overexpression of CHD1L is strongly associated with tumor progression, metastasis, and resistance to therapy 6 , 7 . It regulates DNA repair pathways 39 , 45 , 46 , enhances tumor cell survival, and facilitates chromatin remodeling, allowing cancer cells to evade apoptosis 10 . While its role in tumorigenesis is well-established across various cancers, the function of CHD1L in prostate cancer has not been fully explored. Our study is the first to demonstrate that CHD1L is overexpressed in prostate cancer tissues, contributing to cancer progression. We further show that CHD1L pharmacologic inhibition suppresses tumor cell proliferation and invasion, suggesting its potential as a therapeutic target in prostate cancer. FOXO3 is a well-characterized tumor suppressor that regulates apoptosis, cell cycle progression, DNA repair, and stress responses in various cancers 18 – 20 . In prostate cancer, the loss of FOXO3 function, particularly in androgen-independent forms of the disease, promotes tumor progression and resistance to therapy 27 , 47 . Reactivating FOXO3 has been demonstrated to induce apoptosis and reduce tumor growth 48 – 50 ,including in prostate cancer 51 – 53 . Our study links CHD1L pharmacologic inhibition to FOXO3 activation and identifies the FOXO3-PUMA axis as a key mediator of OTI-611-induced apoptosis. This novel mechanism highlights the therapeutic potential of targeting the FOXO3-PUMA pathway in prostate cancer. Moreover, the combination of OTI-611 with docetaxel resulted in a synergistic enhancement of apoptotic cell death in prostate cancer cells. Docetaxel-based therapies remain a cornerstone for treating metastatic castration-resistant prostate cancer (mCRPC) 54 . However, resistance to docetaxel inevitably develops in most patients, emphasizing the need for combination therapies to overcome this challenge 5 . Our findings provide a strong rationale for combining OTI-611 with docetaxel, as CHD1L inhibition sensitizes prostate cancer cells to chemotherapy-induced apoptosis, potentially overcoming docetaxel resistance and improving clinical outcomes. In conclusion, our study establishes CHD1L as a key oncogenic driver in prostate cancer and uncovers the FOXO3-PUMA axis as a critical mechanism through which CHD1L inhibition induces apoptosis. Furthermore, the synergistic effect of OTI-611 with docetaxel supports its potential as a novel therapeutic approach for advanced prostate cancer. These findings pave the way for targeted therapies that inhibit CHD1L, enhancing the efficacy of existing treatments and addressing the challenges of chemoresistance. Conclusion In summary, our study identifies CHD1L as a critical oncogenic driver in prostate cancer progression. Genetic silencing of CHD1L markedly suppressed cell proliferation and induced apoptosis, while pharmacological inhibition with the selective small-molecule OTI-611 recapitulated these effects both in vitro and in vivo. Mechanistically, OTI-611 activated the FOXO3–PUMA apoptotic axis, and functional suppression of FOXO3 abrogated its antitumor activity, underscoring the pathway’s central role. Furthermore, the combination of OTI-611 with docetaxel produced synergistic antitumor effects, highlighting a potential strategy to enhance the efficacy of current chemotherapy regimens. Together, these findings support CHD1L as a therapeutic vulnerability in prostate cancer and establish OTI-611 as a promising candidate for the development of novel treatment strategies, particularly for advanced or treatment-resistant disease. Abbreviations PCa: Prostate cancer mCRPC: Metastatic castration-resistant prostate cancer CHD1L: Chromodomain helicase DNA-binding protein 1-like FOXO3: Forkhead box O3 BBC3: BCL2 binding component 3 TCGA: The Cancer Genome Atlas GEO: Gene Expression Omnibus IHC: Immunohistochemistry DFS: Disease-free survival RNA-seq: RNA sequencing DEGs: Differential expressed genes Declarations Data availability Supplemental Table is available in the Supplemental Table file. RNA-seq data were deposited in the National Center for Biotechnology Information GEO database and are available under accession number GSE305661. All other data are available in the article and its supplementary materials and are also available upon request from the corresponding author. Acknowledgements None. Funding This study is sponsored by Natural Science Foundation of Chongqing, China (No. CSTB2024NSCQ-MSX1266) and High-level Medical Reserved Personnel Training Project of Chongqing. Author information Authors and Affiliations Department of Urology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China Pusheng Hui & Youlin Kuang School of Basic Medical Sciences, Chongqing Medical University, Chongqing, China Yanru Lai, Haiqi Fan, Qinrong Yan, Zhe Chen & Yu Hou Department of Neurology, the First Affiliated Hospital of Chongqing Medical University, Chongqing, China. Yue Yang Contributions Y. Hou, Z. Chen, and Y. Kuang conceived the project and revised the paper. P. Hui performed experiments, analyzed data, and wrote the paper. Y. Lai, H. Fan, Q. Yan, Y. Yang contributed to the data analysis and paper revision. All authors read and approved the final manuscript. Corresponding authors Correspondence to Zhe Chen, Yu Hou or Youlin Kuang. Ethics declarations Ethical approval The authors affirm that all methods were conducted in accordance with the relevant guidelines and regulations. All mice were housed at the Animal Center of Chongqing Medical University, and approval was obtained from the Animal Committee of the Institute of Zoology at Chongqing Medical University (reference number: IACUC-CQMU-2025-03112, “Mechanism of targeting CHD1L in prostate cancer”). Patient samples were obtained from PCa patients after informed consent of sample use for research. The use of human samples was approved by the Medical Ethics Committees of The First Affiliated Hospital of Chongqing Medical University (reference number: 2025-406-01, “Mechanism of targeting CHD1L in prostate cancer”). Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Additional information Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References Siegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. CA Cancer J Clin. 2024;74:12–49. 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ALC1 links chromatin accessibility to PARP inhibitor response in homologous recombination-deficient cells. Nat Cell Biol. 2021;23:160–71. Singh HR, Nardozza AP, Möller IR, Knobloch G, Kistemaker HAV, Hassler M, et al. A Poly-ADP-Ribose Trigger Releases the Auto-Inhibition of a Chromatin Remodeling Oncogene. Mol Cell. 2017;68:860–e8717. Chen L, Chan THM, Yuan Y-F, Hu L, Huang J, Ma S, et al. CHD1L promotes hepatocellular carcinoma progression and metastasis in mice and is associated with these processes in human patients. J Clin Invest. 2010;120:1178–91. Wang W, Wu J, Fei X, Chen W, Li Y, Shen K, et al. CHD1L promotes cell cycle progression and cell motility by up-regulating MDM2 in breast cancer. Am J Transl Res. 2019;11:1581–92. Liu C, Fu X, Zhong Z, Zhang J, Mou H, Wu Q, et al. CHD1L Expression Increases Tumor Progression and Acts as a Predictive Biomarker for Poor Prognosis in Pancreatic Cancer. Dig Dis Sci. 2017;62:2376–85. Li Y, He L-R, Gao Y, Zhou N-N, Liu Y, Zhou X-K, et al. 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Supplementary Files SupplementaryTable.docx Cite Share Download PDF Status: Published Journal Publication published 04 Dec, 2025 Read the published version in Journal of Translational Medicine → Version 1 posted Reviewers agreed at journal 26 Aug, 2025 Reviewers invited by journal 26 Aug, 2025 Editor assigned by journal 25 Aug, 2025 First submitted to journal 21 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7424447","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":505872079,"identity":"2ec6672c-581f-42ba-a9d9-06deeb219cc9","order_by":0,"name":"Pusheng Hui","email":"","orcid":"","institution":"The First Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Pusheng","middleName":"","lastName":"Hui","suffix":""},{"id":505872080,"identity":"4c34310a-6df5-456d-8d43-ad090161d713","order_by":1,"name":"Yanru Lai","email":"","orcid":"","institution":"Chongqing Medical 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09:18:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7424447/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7424447/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12967-025-07529-5","type":"published","date":"2025-12-04T15:57:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90473819,"identity":"e366cc77-4c8b-4458-a54b-3eaf8a966131","added_by":"auto","created_at":"2025-09-03 06:39:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":284540,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCHD1L is significantly upregulated in prostate cancer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) CHD1L expression in tumor and normal prostate tissues from the TCGA-PRAD cohort.\u003c/p\u003e\n\u003cp\u003e(C-D) Validation of CHD1L expression in PCa datasets GSE69545 and GSE72220.\u003c/p\u003e\n\u003cp\u003e(E) CHD1L expression in AR-independent vs AR-dependent prostate cancer in the GSE2443 dataset.\u003c/p\u003e\n\u003cp\u003e(F) Immunohistochemical staining of CHD1L in prostate cancer tissues from the Protein Atlas database.\u003c/p\u003e\n\u003cp\u003e(G) Kaplan-Meier survival analysis showing disease-free survival (DFS) based on CHD1L expression.\u003c/p\u003e\n\u003cp\u003e(H) Correlation of CHD1L with MKI67, a proliferation marker.\u003c/p\u003e\n\u003cp\u003e(I-J) CHD1L expression in prostate cancer cell lines (Lncap, 22RV1, PC3, DU145) and normal RWPE1 cells.\u003c/p\u003e\n\u003cp\u003e(K-L) CHD1L mRNA and protein levels in paired clinical prostate cancer specimens.\u003c/p\u003e\n\u003cp\u003eError bars represent means±SD. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ns, no significance; student’s t-test.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7424447/v1/95a88c8933992c235ac8e58e.png"},{"id":90473805,"identity":"33506dad-8ea9-4657-afe5-f221a5340f49","added_by":"auto","created_at":"2025-09-03 06:39:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":395246,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCHD1L is essential for survival and proliferation of PCa cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) RT-qPCR analysis of CHD1L expression in DU145 and PC3 PCa cell lines transduced with indicated shRNA lentivirus (n = 3 samples per group).\u003c/p\u003e\n\u003cp\u003e(B) Representative western blot showing the expression of CHD1L and Tubulin in PCa cells transduced with indicated shRNA lentivirus.\u003c/p\u003e\n\u003cp\u003e(C-D) Growth curves of shRNA lentivirus-transduced PCa cells (n = 3 samples per group).\u003c/p\u003e\n\u003cp\u003e(E-F) Representative images of colonies from shRNA lentivirus-transduced PCa cells. The right graph shows the colony numbers (n = 3 samples per group).\u003c/p\u003e\n\u003cp\u003e(G-J) Representative images of invaded (above) and migrated (below) PCa cells following shRNA lentivirus transduction. The right graphs show the invaded (above) and migrated (below) cell numbers (n = 3 samples per group). Scale bar = 100 μm.\u003c/p\u003e\n\u003cp\u003e(K-L) Representative flow plots of apoptosis in shRNA lentivirus-transduced PCa cells using Annexin V/DAPI staining. The graphs indicate the percentages of Annexin V+ cells (n = 3 samples per group).\u003c/p\u003e\n\u003cp\u003eError bars represent means±SD. **p\u0026lt;0.01, ***p\u0026lt;0.001, ns, no significance; one-way ANOVA.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7424447/v1/f971617eb1aa0e3a63003a12.png"},{"id":90473795,"identity":"a04a00ae-bb7c-4f4a-953f-18ff0b91030b","added_by":"auto","created_at":"2025-09-03 06:39:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":372025,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePharmacologic inhibition of CHD1L by OTI-611 suppresses proliferation and induces apoptosis in PCa cells \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) Two-dimensional structure of OTI-611.\u003c/p\u003e\n\u003cp\u003e(B) Composite IC\u003csub\u003e50\u003c/sub\u003e curves in two PCa cell lines following 24-hour treatment with OTI-611, analyzed using the CCK-8 assay.\u003c/p\u003e\n\u003cp\u003e(C-D) Growth curves of PCa cells under treatment conditions with vehicle (DMSO) or 3 μM OTI-611 (n = 3 samples per group).\u003c/p\u003e\n\u003cp\u003e(E-F) Representative images of colonies from PCa cells treated with vehicle or 3 μM OTI-611. The right graph shows the colony numbers (n = 3 samples per group).\u003c/p\u003e\n\u003cp\u003e(G-J) Representative images of invaded (above) and migrated (below) PCa cells treated with vehicle or 3 μM OTI-611. The right graphs show the invaded (above) and migrated (below) cell numbers (n = 3 samples per group). Scale bar = 100 μm.\u003c/p\u003e\n\u003cp\u003e(K-L) Representative flow plots of apoptosis in PCa cells using Annexin V/DAPI staining. DU145 and PC3 were treated with vehicle or 5 μM OTI-611 for 24 h. The graphs show the percentages of Annexin V\u003csup\u003e+\u003c/sup\u003e cells (n = 3 samples per group).\u003c/p\u003e\n\u003cp\u003e(M-N) Representative flow plots of cell cycle of PCa cells using DAPI staining. DU145 and PC3 were treated with vehicle or 5 μM OTI-611 for 24 h. The graphs show the cell cycle distribution (n = 3 samples per group).\u003c/p\u003e\n\u003cp\u003e(O) Representative western blot showing the expression of cell cycle or apoptosis-related proteins and Tubulin in DU145 and PC3 cells treated with vehicle or 5 μM OTI-611 for 24 h\u003c/p\u003e\n\u003cp\u003e(P) Representative western blot shows the expression of CHD1L and Tubulin in PC3 cells transduced with either control or CHD1L overexpression retrovirus (OE-CHD1L).\u003c/p\u003e\n\u003cp\u003e(Q) The growth curves of control and CHD1L-overexpressed PC3 cells treated with vehicle or 3 μM OTI-611 (n = 3 samples per group).\u003c/p\u003e\n\u003cp\u003e(R-S) Representative flow plots of apoptosis in PC3 cells using Annexin V/DAPI staining. Control and CHD1L-overexpressed PC3 cells were treated with vehicle or 5 μM OTI-611 for 24 h. The graphs show the percentages of Annexin V\u003csup\u003e+\u003c/sup\u003e cells (n = 3 samples per group).\u003c/p\u003e\n\u003cp\u003eError bars represent means±SD. **p\u0026lt;0.01, ***p \u0026lt; 0.001, ns, no significance; student’s t-test .\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7424447/v1/0fa79c9e595450fde09d3be5.png"},{"id":90474399,"identity":"4bf367f7-bb81-4017-81d5-8ed3a779c178","added_by":"auto","created_at":"2025-09-03 06:47:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":571760,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOTI-611 impairs the progression of PCa \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) Experimental schematic of the xeno-transplanted mice. Approximately 2×10\u003csup\u003e6\u003c/sup\u003e PC3 cells were subcutaneously injected into the flanks of BALB/c nude mice (day 0). At day 10, mice were treated with either vehicle (DMSO) or OTI-611 (20 mg/kg) every alternate day for a total of 8 days. The mice were euthanized for analysis on day 20.\u003c/p\u003e\n\u003cp\u003e(B-C) Visual comparison of tumors from the mice (n = 5 mice per group).\u003c/p\u003e\n\u003cp\u003e(D) Tumor volume changes in mice (n = 5 mice per group).\u003c/p\u003e\n\u003cp\u003e(E) Tumor weight in different groups of mice (n = 5 mice per group).\u003c/p\u003e\n\u003cp\u003e(F-H) Representative H\u0026amp;E staining, Ki-67 immunofluorescence staining (red), and TUNEL immunofluorescence staining (green) images of tumor in different groups. Nuclei were visualized using DAPI staining (blue). Scale bar, 100 μm.\u003c/p\u003e\n\u003cp\u003e(I) Representative H\u0026amp;E staining images of heart, liver, spleen, lung, and kidney in different groups. Scale bar = 100 μm.\u003c/p\u003e\n\u003cp\u003eError bars represent means±SD. ***p\u0026lt;0.001, ns, no significance; student’s t-test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7424447/v1/62b1d5638733749abe065ac7.png"},{"id":90473833,"identity":"f867a3ad-679f-4658-80f2-4a4e6826fbe7","added_by":"auto","created_at":"2025-09-03 06:39:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":244144,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOTI-611 induces FOXO3/PUMA activation and transcriptional reprogramming in prostate cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) A representative volcano plot of the gene expression change between vehicle- and OTI-611-treated DU145 cells (n = 3 samples per group). All significantly changed genes (\u0026gt; 1 or \u0026lt; -1 log\u003csub\u003e2\u003c/sub\u003e fold change, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) are indicated by blue or red dots.\u003c/p\u003e\n\u003cp\u003e(B-C)\u0026nbsp;Enrichment analysis of DEGs using DAVID and Metascape.\u003c/p\u003e\n\u003cp\u003e(D) GSEA shows significantly regulated signaling in OTI-611-treated PC3 cells compared to vehicle-treated cells.\u003c/p\u003e\n\u003cp\u003e(E) RT-qPCR analysis of FOXO3 and PUMA expression in PCa cell lines. DU145 and PC3 cells were treated with vehicle or 5 μM OTI-611 for 24 h (n = 3 samples per group).\u003c/p\u003e\n\u003cp\u003e(F) Representative western blot showing the expression of FOXO3, PUMA and Tubulin of PCa cells after treatment with vehicle or OTI-611.\u003c/p\u003e\n\u003cp\u003eError bars represent means±SD. ***p\u0026lt;0.001, ns, no significance; student’s t-test.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7424447/v1/aae405e5bfc4e2c01eec7978.png"},{"id":90473796,"identity":"7057a748-2efd-40ce-8495-616956805db7","added_by":"auto","created_at":"2025-09-03 06:39:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":247627,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFOXO3 knockdown reduces the anti-tumor effects of OTI-611\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) RT-qPCR analysis of FOXO3 expression in PCa cell lines transduced with three independent siRNAs (n = 3 samples per group).\u003c/p\u003e\n\u003cp\u003e(C-D) Representative western blot showing the expression of FOXO3 and Tubulin in PCa cell lines transduced with three independent siRNAs.\u003c/p\u003e\n\u003cp\u003e(E-F) Growth curves of control and FOXO3-knockdown PCa cells under treatment conditions with vehicle or 3 μM OTI-611 (n = 3 samples per group)\u003c/p\u003e\n\u003cp\u003e(G-H) Representative flow plots showing apoptosis in PCa cells using Annexin V/DAPI staining.Control and FOXO3-knockdown PCa cells were treated with vehicle or 5 μM OTI-611 for 24 h. The graphs show the percentages of Annexin V+ cells (n = 3 samples per group).\u003c/p\u003e\n\u003cp\u003e(I) Representative western blot showing the expression of PUMA and Tubulin in PCa cell lines transduced with three independent siRNAs\u003c/p\u003e\n\u003cp\u003e(J) Representative western blot showing the expression of PUMA and Tubulin in control and FOXO3-knockdown PCa cells treated with vehicle or 5 μM OTI-611 for 24 h\u003c/p\u003e\n\u003cp\u003eError bars represent means±SD. **p\u0026lt;0.01, ***p\u0026lt;0.001, ns, no significance; one-way ANOVA.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7424447/v1/885398be02c0d721bac454bf.png"},{"id":90473829,"identity":"6e567940-7015-4b16-aeba-29acf5e8a385","added_by":"auto","created_at":"2025-09-03 06:39:44","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":222038,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of CHD1L by OTI-611 sensitizes PCa cells to docetaxel\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) Inhibition rate of PCa cells using the CCK-8 assay. PCa cells were treated with varying doses of OTI-611, docetaxel, or a combination of both for a duration of 48 h (3 samples per condition).\u003c/p\u003e\n\u003cp\u003e(C-D) Synergy score analysis was conducted using the web application SynergyFinder (\u003ca href=\"https://synergyfinder.fimm.fi\"\u003ehttps://synergyfinder.fimm.fi\u003c/a\u003e).\u003c/p\u003e\n\u003cp\u003e(E-F) Representative flow plots showing apoptosis in PCa cells using Annexin V/DAPI staining. PCa cells were treated with 3 μM OTI-611, 10 nM docetaxel or a combination of both for 48 h. The graphs show the percentages of Annexin V\u003csup\u003e+\u003c/sup\u003e cells (n = 3 samples per group).\u003c/p\u003e\n\u003cp\u003eError bars represent means±SD. ***p\u0026lt;0.001, ns, no significance; one-way ANOVA.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7424447/v1/ca9de5b1e56a0d77b795c1dc.png"},{"id":97723939,"identity":"319baac1-4b27-4b2f-a7c2-5f01d1687c3a","added_by":"auto","created_at":"2025-12-08 16:09:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3338739,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7424447/v1/b7c8aab2-6ba9-41f5-96f1-eb4e7e8f299a.pdf"},{"id":90473821,"identity":"4c8a7990-0736-4f5b-aa89-f1c9e093eb30","added_by":"auto","created_at":"2025-09-03 06:39:43","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":17732,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-7424447/v1/1667be9f6c72e997ea239b73.docx"}],"financialInterests":"","formattedTitle":"Targeting CHD1L suppresses prostate cancer progression via the FOXO3-PUMA axis","fulltext":[{"header":"Background","content":"\u003cp\u003eProstate cancer (PCa) is the most common malignancy among men globally\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Although androgen deprivation therapy (ADT) is initially effective, most patients with advanced disease progress to castration-resistant prostate cancer (CRPC), where treatment options are limited and prognosis is poor\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Taxane-based chemotherapy, particularly docetaxel, improves survival in both metastatic hormone-sensitive and castration-resistant settings\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. However, approximately 50% of patients initially respond to docetaxel, but nearly all develop resistance within 6\u0026ndash;8 months of treatment\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, highlighting the need for novel therapeutic strategies and effective combination regimens.\u003c/p\u003e\u003cp\u003eChromodomain helicase DNA-binding protein 1-like (CHD1L) is an ATP-dependent chromatin remodeler that regulates DNA repair, transcription, and cell survival pathways\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Aberrant CHD1L expression is implicated in several malignancies, where it promotes proliferation, metastasis, and therapy resistance\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Despite its well-established role in other cancers, the clinical significance and therapeutic potential of CHD1L in prostate cancer remain underexplored. OTI-611 is a small-molecule inhibitor of CHD1L that binds its ATPase/helicase domain, displaying potent antitumor activity through allosteric inhibition of CHD1L ATPase\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Although OTI-611 has demonstrated anti-tumor activity in other malignancies\u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, its efficacy and underlying mechanisms in prostate cancer remain to be fully elucidated. Forkhead Box O3 (FOXO3), a key tumor suppressor transcription factor, regulates apoptosis, cell cycle arrest, DNA damage repair, and stress responses\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Its pro-apoptotic target, PUMA (BBC3), is a potent BH3-only protein that triggers apoptosis\u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Activation of the FOXO3-PUMA axis has been shown to induce robust apoptosis in prostate cancer cells\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, making it an attractive therapeutic target.\u003c/p\u003e\u003cp\u003eHere, we demonstrate that both knockdown and pharmacological inhibition of CHD1L using OTI-611 induce apoptosis in prostate cancer cells \u003cem\u003ein vitro\u003c/em\u003e and inhibit tumor progression \u003cem\u003ein vivo\u003c/em\u003e. Mechanistically, we identify the FOXO3-PUMA axis as a key mediator of OTI-611-induced apoptosis. Furthermore, we show that OTI-611 synergizes with docetaxel to enhance apoptotic cell death, providing a strong rationale for its use in combination therapy for advanced prostate cancer.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePatient samples and cell culture\u003c/h2\u003e\u003cp\u003eAll samples used in this study were acquired from 6 patients with PCa at The First Affiliated Hospital of Chongqing Medical University. This study was conducted under the guidelines of the Declaration of Helsinki and was approved by the Institutional Ethics Committee.\u003c/p\u003e\u003cp\u003eThe HEK293T cell line was obtained from the American TypeCulture Collection (ATCC). The prostate cancer cell lines DU145, PC3 ,22RV1 and LnCAP and the non-malignant prostate epithelial cell line RWPE1 were all obtained from Procell Life Science \u0026amp; Technology. Cell lines were reauthenticated through short tandem repeat analysis and assessed for mycoplasma contamination every six months following thawing in our experiments. DU145, PC3 ,22RV1 and LnCAP cells were cultured in MEM or RPMI 1640 medium supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Hyclone). The RWPE-1 cell line was cultured in K-SFM. HEK293T cells were maintained in DMEM containing 10% FBS and 1% penicillin/streptomycin.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eChemical compounds\u003c/h3\u003e\n\u003cp\u003eFor \u003cem\u003ein vitro\u003c/em\u003e studies, OTI-611(CHD1Li 6.11, #HY-144256,MCE), and Docetaxel (#HY-B0011 ,MCE) were dissolved in DMSO to stock concentrations of 10mM. For \u003cem\u003ein vivo\u003c/em\u003e experiments, OTI-611 was dissolved in 2% DMSO, 40%PEG300, 5% Tween-80, and 53% saline.\u003c/p\u003e\n\u003ch3\u003ePlasmids and lentivirus\u003c/h3\u003e\n\u003cp\u003eTo generate the vectors for the expression of CHD1L-specific shRNA, we designed the sequence of shRNAs and cloned shRNAs into the vector pLKO.1-GFP-puro (refer to primer sequences in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). To generate the vectors for the expression of human CHD1L, the coding sequences were synthesized by Tsingke (China) and cloned into the vector pMSCV-IRES-EGFP. Small interfering RNAs (siRNAs) targeted FOXO3 were purchased from Tsingke (China) (refer to siRNA sequences in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). For virus production, HEK293T was transfected with lentiviral or retroviral plasmid with helper plasmid (psPAX2 and pMD.2G together with pLKO-shRNA vectors, pCL-Ampho together with pMSCV vectors). The medium was replaced with fresh medium at 12 h after transfection and culture supernatants were collected at 48 h and 72 h. The virus was stored at -80\u0026deg;C until use. Positively infected cells were isolated using flow cytometry sorting (GFP+) or puromycin treatment.\u003c/p\u003e\n\u003ch3\u003eReal-time quantitative reverse transcription PCR (RT-qPCR)\u003c/h3\u003e\n\u003cp\u003eThe RT-qPCR was performed as described previously\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Generally, total RNA was isolated using the Total RNA Isolation Kit (Thermo Fisher) according to the manufacturer\u0026rsquo;s instructions. cDNA was reversetranscribed using PrimeScript RT reagent Kit (Takara) and subjected to real-time PCR with SYBR Green Supermix (Bio-Rad) in an iCycler iQ Real- Time PCR Detection System (Bio-Rad). All primers are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. All samples were run in triplicate. GAPDH was used as an internal control for mRNA.\u003c/p\u003e\n\u003ch3\u003eCell proliferation and colony formation assays\u003c/h3\u003e\n\u003cp\u003eCell proliferation was assessed using CCK8 kit (MA0218-5,Meilunbio) according to the manufacturer\u0026rsquo;s instructions. Cells (2\u0026times; 10\u003csup\u003e3\u003c/sup\u003e) suspended in 100 \u0026micro;l medium were seeded in triplicate in a 96-well plate, grown at 37\u0026deg;C. Medium containing 10% CCK8 replaced the original medium and incubated at 37\u0026deg;C for 2\u0026ndash;4 h, and the absorbance was finally determined at 450 nm using a micro plate reader.\u003c/p\u003e\u003cp\u003eFor colony formation assay, 1 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells were cultured in 6-well plate at 37\u0026deg;C for 10\u0026ndash;14 days, visible colonies were washed twice with phosphate-buffered saline (PBS), then fixed with 4% paraformaldehyde for 30 min following a suitable incubation period and subsequently stained with crystal violet. The number of colonies was counted visually.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eFlow cytometry analysis\u003c/h2\u003e\u003cp\u003eFor the cell cycle analysis, 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e prostate cancer cells were washed with PBS and then fixed in 70% cold ethanol at 4\u0026deg;C overnight. Then, the cells were stained with DAPI (Biolegend, 422801) / RNase mixture in the dark at 37\u0026deg;C for 1 h. For the apoptosis assay, 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e prostate cancer cells were harvested, washed, and suspended in a binding buffer containing Annexin V-PE (Biolegend, 640947) and DAPI. All samples were analyzed by Flow cytometry on a BD FACS Canto II system, and all data were analyzed using FlowJo software.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eWestern blot\u003c/h3\u003e\n\u003cp\u003eFor the analysis of cell proteins, prostate cancer cells were lysed using RIPA lysis buffer (Beyotime). The resulting protein extracts were then separated by SDS-PAGE. The antibodies used in this study were against the following: rabbit anti-CHD1L(1:1000; #32923-1-AP; Proteintech), rabbit anti-Cyclin B1 (1:1000; #4138; CST), rabbit anti-Cyclin E1 (1:1000; #20808; CST), rabbit anti-Cdk1 (1:1000; #ET1607-51; HUABIO), rabbit anti-Cdk2 (1:1000; #ET1602-6; HUABIO), rabbit anti-BAX (1:1000; #2772; CST), rabbit anti-BCL-2 (1:1000; #3498; CST), rabbit anti-FOXO3 (1:1000; #2497; CST), rabbit anti-PUMA (1:1000; #98672; CST), rabbit anti-Tubulin (1:2000; #AF001; Beyotime). Secondary antibodies was HRP-conjugated Goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) (1:2500; #A0208;Beyotime).\u003c/p\u003e\n\u003ch3\u003eCell migration and invasion assay\u003c/h3\u003e\n\u003cp\u003eApproximately 2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e PCa cells were resuspended in 100 \u0026micro;L serum-free medium. Then the cells were inoculated into the upper transwell chamber of the insert (8-mm pores), and 600 \u0026micro;L of complete medium containing 20% FBS was added to the lower chamber. After 24h migration, the cells in low chambers were fixed with 4% paraformaldehyde for 30 min following a suitable incubation period and subsequently stained with crystal violet. Finally, random fields were imaged using an inverted microscope at 100\u0026times; magnification. The number of cells in each image was calculated using the ImageJ software. Cell invasion studies were performed according to a similar Transwell method described above, except the upper chamber was pre-coated with Matrigel (356234,Corning).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eTransplantation of xenogeneic tumor cells\u003c/h2\u003e\u003cp\u003eApproximately 2\u0026times; 10\u003csup\u003e6\u003c/sup\u003e PC3 cells were subcutaneously injected into the flanks of BALB/c nude mice (male, 8\u0026ndash;10 weeks old) (day 0). After 10 days, mice were treated with vehicle or OTI-611 (20 mg/kg) every alternate day for a total of 8 days (days 10\u0026ndash;18). Tumor volume was measured using vernier calipers every 2 days (days 10\u0026ndash;20) and calculated with the formula: V\u0026thinsp;=\u0026thinsp;0.5 \u0026times; L \u0026times; W\u003csup\u003e2\u003c/sup\u003e, where L is the longest diameter of the tumor and W is the longest transverse diameter perpendicular to L. On day 20, the mice were euthanized and the weights of the tumors were measured. The isolated tissues were immersed in 4% polyformaldehyde and sectioned at Aifang Biotechnology Co., Ltd. Tumor sections obtained from each group were stained with hematoxylin and eosin (H\u0026amp;E) (BP092; Biossci, Wuhan, China) to identify features of the xenograft tumors. Ki-67 (ab92742; Abcam, Cambridge, UK) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) (A112-03; Vazyme, Nanjing, China) were used to assess proliferation and apoptosis, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eRNA-sequencing and data analysis\u003c/h2\u003e\u003cp\u003eTotal RNA samples were extracted from DU145 cells upon OTI-611 treatment. RNA-seq was performed by Sangon Science (Shanghai, China), and the libraries were sequenced by the Illumina HiSeq 2000 platform as 150-bp pair-ended reads. RNA-seq data were analyzed according to previous work\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Briefly, Fragments per kilobase per million (FPKM) estimation was performed with Cufflinks v2.1.1, aligned reads were counted with HTSeq (a Python framework to work with high-throughput sequencing data), and differential expression analysis was performed with DESeq2. Differentially expressed genes were identified based on a log2 fold change greater than 1 or less than \u0026minus;\u0026thinsp;1, accompanied by statistics. The volcano plot illustrating gene expression was generated using R. GO enrichment and pathway analyses was performed using the DAVID (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://david.ncifcrf.gov/tools.jsp\u003c/span\u003e\u003cspan address=\"https://david.ncifcrf.gov/tools.jsp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e56\u003c/sup\u003e and Metascape database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://metascape.org\u003c/span\u003e\u003cspan address=\"http://metascape.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e57\u003c/sup\u003e. Gene set enrichment analysis was performed using GSEA software (v4.1.0) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.broadinstitute.org/gsea\u003c/span\u003e\u003cspan address=\"http://www.broadinstitute.org/gsea\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with 1000 permutations.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eSynergy score analysis\u003c/h2\u003e\u003cp\u003eThe synergy score analysis was conducted using the web application SynergyFinder (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://synergyfinder.fimm.fi\u003c/span\u003e\u003cspan address=\"https://synergyfinder.fimm.fi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e58\u003c/sup\u003e. The synergy score was determined by assessing the deviation from the theoretical inhibition calculated using the HSA method. According to the user guide available on the website, the synergy score is assessed as follows: scores less than \u0026minus;\u0026thinsp;10 indicate that the interaction between two drugs is likely to be antagonistic; scores ranging from \u0026minus;\u0026thinsp;10 to 10 suggest that the interaction is likely to be additive; and scores greater than 10 imply that the interaction is likely to be synergistic.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eStatistics analysis\u003c/h2\u003e\u003cp\u003eAll experiments were repeated at least two times independently, and at least three independently samples were used for data analysis. We used simple randomization to allocate samples/animals to experimental groups. The sample size was not pre-selected, and no samples or animals were excluded from the analysis. Single-blind blinding was implemented in the experiment. The investigator was blinded to the group allocation during the outcome assessment. All statistical analysis was performed using GraphPad Prism 8 software (GraphPad Software). Statistical significance between the two groups was analyzed using a two-tailed unpaired Student\u0026rsquo;s t-test. For metaphase analysis, statistical comparisons were made using a one-way ANOVA test followed by Tukey\u0026ndash;Kramer post-hoc tests. All data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). P values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eCHD1L is upregulated in prostate cancer and correlates with poor prognosis\u003c/h2\u003e\u003cp\u003eTo elucidate the potential role of CHD1L in prostate cancer (PRAD), we comprehensively assessed its expression across multiple publicly available datasets. In the TCGA-PRAD cohort, CHD1L was markedly upregulated in tumor tissues compared with normal prostate tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). These findings were consistently validated in independent GEO datasets, including GSE69545 and GSE72220 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D). Analysis of the GSE2443 dataset revealed markedly higher CHD1L expression in AR-independent prostate cancer, an aggressive subtype with poorer prognosis\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, compared with AR-dependent tumors (Figure. 1E), suggesting that CHD1L upregulation may contribute to androgen receptor-independent disease progression. Moreover, immunohistochemical (IHC) staining from the Protein Atlas database revealed moderate CHD1L staining in 16 of 20 PRAD samples, whereas all three normal samples displayed undetectable expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Clinically, elevated CHD1L expression was associated with significantly poorer disease-free survival (DFS), as demonstrated by Kaplan-Meier analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Moreover, CHD1L expression was positively correlated with MKI67, a well-established proliferation marker (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH), implicating CHD1L in tumor growth progression. Consistently, increased CHD1L expression was observed at both mRNA and protein levels in prostate cancer cell lines (22RV1, PC3, and DU145) compared with the normal prostate epithelial cell line RWPE1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI-J). Paired clinical specimens further demonstrated significantly elevated CHD1L mRNA and protein levels in tumor tissues relative to adjacent normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK-L). Collectively, these data demonstrate that CHD1L is frequently overexpressed in prostate cancer and may link to tumor progression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eKnockdown of CHD1L suppresses proliferation, migration, invasion, and promotes apoptosis in prostate cancer cells\u003c/h2\u003e\u003cp\u003eTo investigate the functional role of CHD1L in prostate cancer, we silenced CHD1L using two independent shRNAs in DU145 and PC3 cells. RT-qPCR and western blotting confirmed efficient CHD1L knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). CCK-8 and colony formation assays demonstrated that CHD1L depletion markedly suppressed cell growth and clonogenic capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-F). Transwell assays further revealed that CHD1L knockdown significantly impaired both migration and invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-J). Moreover, Annexin V/DAPI flow cytometry showed increased apoptosis following CHD1L silencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK-L). Collectively, these data identify CHD1L as a critical oncogenic driver that promotes proliferation, migration, invasion, and survival of prostate cancer cells \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eOTI-611, a small molecule inhibitor of CHD1L, suppresses tumor cell growth and induces apoptosis in prostate cancer\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess the effects of pharmacologic CHD1L inhibition, we treated DU145 and PC3 cells with OTI-611 (CHD1Li 6.11), a small molecule inhibitor that binds to the ATPase/helicase domain of CHD1L\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Dose-response analysis revealed that OTI-611 reduced cell viability in both cell lines, with IC\u003csub\u003e50\u003c/sub\u003e values of 4.380 \u0026micro;M and 3.963 \u0026micro;M, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). 3 \u0026micro;M OTI-611 treatment significantly suppressed proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D) and decreased clonogenic capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F). Transwell assays demonstrated that OTI-611 significantly impaired migration and invasion in both DU145 and PC3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-J). Annexin V and DAPI staining revealed a robust increase in apoptotic cell populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK-L). It has been reported that OTI-611 impairs the cell-cycle progression in breast and colorectal cancer\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. In this study, we observed that OTI-611 treatment led to an increased percentage of PCa cells in G2/M phase, associated with a reduced percentage of cells in G0/G1 phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM-N). Mechanistically, western blot analysis revealed that OTI-611 treatment resulted in changes of Cyclin B1, Cyclin E1, CDK1, and CDK2, along with increased expression of pro-apoptotic proteins BAX and reduced expression of the anti-apoptotic protein BCL2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eO), further supporting the functional role of CHD1L in regulating cell cycle progression and apoptosis. To validate the specificity of OTI-611, we overexpressed CHD1L in PC3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eP) and found that CHD1L overexpression partially rescued the OTI-611-induced growth suppression and apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eQ-S), confirming that the observed phenotypes are CHD1L-dependent.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTogether, these findings suggest that the pharmacologic inhibition of CHD1L by OTI-611 efficiently inhibits proliferation, migration and invasion and induces apoptosis of PCa cells \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOTI-611 suppresses tumor growth\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e \u003cb\u003ewithout inducing systemic toxicity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe next assessed the \u003cem\u003ein vivo\u003c/em\u003e efficacy of OTI-611 using a PC3 xenograft model in BALB/c nude mice. Ten days after tumor implantation, mice were randomly assigned to receive intraperitoneal injections of OTI-611 (20 mg/kg)\u003csup\u003e11\u003c/sup\u003e or vehicle every other day for 8 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). As the treatment progressed, OTI-611 markedly suppressed tumor growth, leading to significant reductions in tumor volume and weight compared with controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-E). Histological examination further supported these findings. H\u0026amp;E staining of tumor sections revealed marked cell disintegration in the OTI-611 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). In addition, Ki67 immunostaining showed a pronounced decrease in proliferative activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG), indicating reduced tumor cell growth \u003cem\u003ein vivo\u003c/em\u003e. Consistent with our \u003cem\u003ein vitro\u003c/em\u003e results, TUNEL staining demonstrated increased apoptotic cells in tumors from OTI-611-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). To evaluate safety, we performed histopathological analyses of major organs, including the heart, liver, spleen, lung, and kidney. No detectable abnormalities were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI), suggesting that OTI-611 is well tolerated at the therapeutic dose. Taken together, these results confirm that pharmacologic CHD1L inhibition by OTI-611 effectively suppresses prostate cancer growth \u003cem\u003ein vivo\u003c/em\u003e while exhibiting minimal systemic toxicity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eOTI-611 activates FOXO3-mediated apoptotic signaling pathway in prostate cancer cells\u003c/h2\u003e\u003cp\u003eTo investigate the molecular mechanisms underlying the anti-tumor effects of CHD1L inhibition, RNA sequencing was performed on DU145 cells treated with OTI-611 or vehicle, identifying 537 upregulated and 673 downregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). GO enrichment and pathway analyses indicated significant activation of transcriptional regulation, chromatin remodeling, DNA damage response, apoptotic signaling, and stress-related processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C). Among these, FOXO3 signaling was prominently enriched (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). FOXO3 is a transcription factor regulating genes involved in apoptosis, proliferation, cell cycle progression, DNA damage repair, stress responses, and tumorigenesis\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In prostate cancer, FOXO3 functions as a tumor suppressor\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, and its activation has been proposed as a therapeutic strategy\u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. PUMA (BBC3), a well-established FOXO3 transcriptional target \u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, was among the most highly upregulated genes after OTI-611 treatment. Previous studies have shown that activation of the FOXO3-PUMA axis induces potent apoptosis in prostate cancer cells\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, underscoring its importance in therapeutic intervention. GSEA further demonstrated enrichment of TNFα signaling via NF-κB, p53 signaling, and unfolded protein response in OTI-611-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Conversely, gene sets related to DNA repair, MYC targets, and interferon alpha response were negatively enriched, suggesting suppression of survival pathways. RT-qPCR confirmed that OTI-611 significantly increased FOXO3 and PUMA expression in DU145 and PC3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Western blotting validated a time- and dose-dependent upregulation of both proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), supporting activation of the FOXO3-mediated apoptotic pathway. Collectively, these findings identify the FOXO3-PUMA axis as a key downstream effector of CHD1L inhibition by OTI-611 in prostate cancer.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eFOXO3 knockdown attenuates the anti-tumor effects of OTI-611 in prostate cancer cells\u003c/h2\u003e\u003cp\u003eTo determine whether FOXO3 is required for the anti-tumor activity of OTI-611, FOXO3 was silenced in DU145 and PC3 cells using three independent siRNAs, with siFOXO3-2 achieving the most efficient knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-D). CCK-8 assays revealed that FOXO3 silencing largely rescued OTI-611-mediated growth inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-F), while Annexin V/DAPI staining showed a marked reduction in OTI-611-induced apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG-H). Western blotting demonstrated that FOXO3 knockdown significantly decreased PUMA expression, and that OTI-611-driven PUMA induction was abolished in FOXO3-deficient cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI-J). These findings identify FOXO3 as a pivotal mediator of OTI-611\u0026rsquo;s anti-cancer effects and highlight the FOXO3-PUMA axis as a central apoptotic pathway downstream of OTI-611 treatment in prostate cancer.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eOTI-611 synergizes with docetaxel to enhance apoptosis in prostate cancer cells\u003c/h2\u003e\u003cp\u003eTaxanes such as docetaxel are among the most active chemotherapies for advanced prostate cancer\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, yet resistance inevitably develops in nearly all patients\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Given that CHD1L acts as a key anti-apoptotic factor in prostate cancer, we hypothesized that its inhibition by OTI-611 could enhance docetaxel efficacy. To test this, DU145 and PC3 cells were treated with escalating doses of docetaxel, OTI-611, or their combination.\u003c/p\u003e\u003cp\u003eCCK-8 assays and heatmaps demonstrated that combination treatment produced greater growth inhibition than either agent alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-B). SynergyFinder analysis confirmed a synergistic interaction (synergy score\u0026thinsp;\u0026gt;\u0026thinsp;10) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-D). Annexin V/DAPI staining further showed that, while each drug alone induced moderate apoptosis, their combination markedly increased apoptotic cell populations in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-H). These findings indicate that OTI-611 synergizes with docetaxel to promote apoptosis in prostate cancer cells, supporting its potential for combination therapy.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eChromatin remodeling factors play a crucial role in tumorigenesis by regulating DNA repair, gene expression, and genomic stability, which are essential for tumor cell survival and proliferation under stress conditions\u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. CHD1L, an ATP-dependent chromatin remodeler\u003csup\u003e\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, has been implicated as an oncogene in multiple malignancies, including liver\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, breast\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, ovarian\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, pancreas\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, and lung cancer\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Overexpression of CHD1L is strongly associated with tumor progression, metastasis, and resistance to therapy\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. It regulates DNA repair pathways\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, enhances tumor cell survival, and facilitates chromatin remodeling, allowing cancer cells to evade apoptosis\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. While its role in tumorigenesis is well-established across various cancers, the function of CHD1L in prostate cancer has not been fully explored. Our study is the first to demonstrate that CHD1L is overexpressed in prostate cancer tissues, contributing to cancer progression. We further show that CHD1L pharmacologic inhibition suppresses tumor cell proliferation and invasion, suggesting its potential as a therapeutic target in prostate cancer.\u003c/p\u003e\u003cp\u003eFOXO3 is a well-characterized tumor suppressor that regulates apoptosis, cell cycle progression, DNA repair, and stress responses in various cancers\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In prostate cancer, the loss of FOXO3 function, particularly in androgen-independent forms of the disease, promotes tumor progression and resistance to therapy\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Reactivating FOXO3 has been demonstrated to induce apoptosis and reduce tumor growth\u003csup\u003e\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e,including in prostate cancer\u003csup\u003e\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Our study links CHD1L pharmacologic inhibition to FOXO3 activation and identifies the FOXO3-PUMA axis as a key mediator of OTI-611-induced apoptosis. This novel mechanism highlights the therapeutic potential of targeting the FOXO3-PUMA pathway in prostate cancer.\u003c/p\u003e\u003cp\u003eMoreover, the combination of OTI-611 with docetaxel resulted in a synergistic enhancement of apoptotic cell death in prostate cancer cells. Docetaxel-based therapies remain a cornerstone for treating metastatic castration-resistant prostate cancer (mCRPC)\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. However, resistance to docetaxel inevitably develops in most patients, emphasizing the need for combination therapies to overcome this challenge\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Our findings provide a strong rationale for combining OTI-611 with docetaxel, as CHD1L inhibition sensitizes prostate cancer cells to chemotherapy-induced apoptosis, potentially overcoming docetaxel resistance and improving clinical outcomes.\u003c/p\u003e\u003cp\u003eIn conclusion, our study establishes CHD1L as a key oncogenic driver in prostate cancer and uncovers the FOXO3-PUMA axis as a critical mechanism through which CHD1L inhibition induces apoptosis. Furthermore, the synergistic effect of OTI-611 with docetaxel supports its potential as a novel therapeutic approach for advanced prostate cancer. These findings pave the way for targeted therapies that inhibit CHD1L, enhancing the efficacy of existing treatments and addressing the challenges of chemoresistance.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, our study identifies CHD1L as a critical oncogenic driver in prostate cancer progression. Genetic silencing of CHD1L markedly suppressed cell proliferation and induced apoptosis, while pharmacological inhibition with the selective small-molecule OTI-611 recapitulated these effects both in vitro and in vivo. Mechanistically, OTI-611 activated the FOXO3\u0026ndash;PUMA apoptotic axis, and functional suppression of FOXO3 abrogated its antitumor activity, underscoring the pathway\u0026rsquo;s central role. Furthermore, the combination of OTI-611 with docetaxel produced synergistic antitumor effects, highlighting a potential strategy to enhance the efficacy of current chemotherapy regimens. Together, these findings support CHD1L as a therapeutic vulnerability in prostate cancer and establish OTI-611 as a promising candidate for the development of novel treatment strategies, particularly for advanced or treatment-resistant disease.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003ePCa:\u003c/strong\u003e Prostate cancer\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emCRPC:\u003c/strong\u003e Metastatic castration-resistant prostate cancer\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCHD1L:\u003c/strong\u003e Chromodomain helicase DNA-binding protein 1-like\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFOXO3:\u003c/strong\u003e Forkhead box O3\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBBC3:\u003c/strong\u003e BCL2 binding component 3\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTCGA:\u003c/strong\u003e The Cancer Genome Atlas\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGEO:\u003c/strong\u003e Gene Expression Omnibus\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIHC:\u003c/strong\u003e Immunohistochemistry\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDFS:\u003c/strong\u003e Disease-free survival\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA-seq:\u003c/strong\u003e RNA sequencing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDEGs:\u003c/strong\u003e Differential expressed genes\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplemental Table is available in the Supplemental Table file.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eRNA-seq data were deposited in the National Center for Biotechnology Information GEO database and are available under accession number GSE305661. All other data are available in the article and its supplementary materials and are also available upon request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is sponsored\u0026nbsp;by\u0026nbsp;Natural\u0026nbsp;Science\u0026nbsp;Foundation\u0026nbsp;of\u0026nbsp;Chongqing, China (No. CSTB2024NSCQ-MSX1266) and High-level Medical Reserved Personnel Training Project of Chongqing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors and Affiliations\u003c/p\u003e\n\u003cp\u003eDepartment of Urology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China\u003c/p\u003e\n\u003cp\u003ePusheng Hui \u0026amp; Youlin Kuang\u003c/p\u003e\n\u003cp\u003eSchool of Basic Medical Sciences, Chongqing Medical University, Chongqing, China\u003c/p\u003e\n\u003cp\u003eYanru Lai, Haiqi Fan, Qinrong Yan, Zhe Chen \u0026amp; Yu Hou\u003c/p\u003e\n\u003cp\u003eDepartment of Neurology, the First Affiliated Hospital of Chongqing Medical University, Chongqing, China.\u003c/p\u003e\n\u003cp\u003eYue Yang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY. Hou, Z. Chen, and Y. Kuang conceived the project and revised the paper. P. Hui performed experiments, analyzed data, and wrote the paper. Y. Lai, H. Fan, Q. Yan, Y. Yang contributed to the data analysis and paper revision. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Zhe Chen, Yu Hou or Youlin Kuang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors affirm that all methods were conducted in accordance with the relevant guidelines and regulations. All mice were housed at the Animal Center of Chongqing Medical University, and approval was obtained from the Animal Committee of the Institute of Zoology at Chongqing Medical University (reference number: IACUC-CQMU-2025-03112, \u0026ldquo;Mechanism of targeting CHD1L in prostate cancer\u0026rdquo;). Patient samples were obtained from PCa patients after informed consent of sample use for research. The use of human samples was approved by the Medical Ethics Committees of The First Affiliated Hospital of Chongqing Medical University\u0026nbsp;(reference number: 2025-406-01, \u0026ldquo;Mechanism of targeting CHD1L in prostate cancer\u0026rdquo;).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePublisher\u0026rsquo;s note\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Giaquinto AN, Jemal A. 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Nucleic Acids Res. 2022;50:W739\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Prostate cancer, CHD1L, FOXO3, PUMA, Docetaxel","lastPublishedDoi":"10.21203/rs.3.rs-7424447/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7424447/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eProstate cancer (PCa) is one of the most common malignancies in men worldwide, and advanced or metastatic disease remains a major therapeutic challenge. Chromodomain helicase DNA binding protein 1-like (CHD1L) has been implicated as an oncogenic driver in multiple cancer types, yet its role in prostate cancer pathogenesis is not fully defined. The purpose of this study is to investigate the biological significance of CHD1L in prostate cancer and to evaluate the therapeutic potential of its selective inhibitor OTI-611.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eBioinformatics analyses were conducted to assess the expression, prognostic significance of CHD1L in PCa patients. \u003cem\u003eIn vitro\u003c/em\u003e, cell viability, cycle progression, apoptosis, and migration/invasion were evaluated using CCK-8, colony formation, flow cytometry, transwell assays. \u003cem\u003eIn vivo\u003c/em\u003e treatment potential of OTI-611 was assessed through a nude mouse xenograft model. Protein and mRNA levels were determined by western blot and qPCR, respectively. Synergism of OTI-611 and docetaxel was determined using SynergyFinder 3.0.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eWe demonstrated that CHD1L was significantly upregulated in PCa patients and correlates with poor prognosis. Genetic knockdown of CHD1L substantially inhibits PCa cell proliferation and induces apoptosis. Moreover, inhibition of CHD1L by the small molecule OTI-611 significantly suppresses PCa cell proliferation, migration, and invasion, and induces apoptosis both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Mechanistically, inhibition of CHD1L induces the expression of FOXO3 (a classic transcription factor) and its downstream target PUMA (a key apoptosis inducer). Restricting the expression of FOXO3 significantly reverses the anti-tumor effects induced by OTI-611. Furthermore, OTI-611 synergizes with docetaxel to enhance apoptotic cell death, providing a promising strategy to overcome docetaxel resistance.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eOur study demonstrates that CHD1L is markedly upregulated in prostate cancer and contributes to tumor progression. Pharmacological inhibition of CHD1L with the selective inhibitor OTI-611 significantly suppresses proliferation, migration, and invasion, while inducing apoptosis \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Mechanistically, these effects are mediated through activation of the FOXO3\u0026ndash;PUMA axis, as FOXO3 suppression abrogates OTI-611\u0026ndash;induced apoptosis. Moreover, OTI-611 exhibits strong synergy with docetaxel, enhancing apoptotic cell death and providing a potential strategy to improve therapeutic efficacy in prostate cancer.\u003c/p\u003e","manuscriptTitle":"Targeting CHD1L suppresses prostate cancer progression via the FOXO3-PUMA axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-03 06:39:26","doi":"10.21203/rs.3.rs-7424447/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-08-26T14:36:15+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-26T13:37:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-25T09:06:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Translational Medicine","date":"2025-08-21T05:18:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ccfe974d-95b3-473f-ab6a-635c27f8b3f3","owner":[],"postedDate":"September 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T16:03:07+00:00","versionOfRecord":{"articleIdentity":"rs-7424447","link":"https://doi.org/10.1186/s12967-025-07529-5","journal":{"identity":"journal-of-translational-medicine","isVorOnly":false,"title":"Journal of Translational Medicine"},"publishedOn":"2025-12-04 15:57:19","publishedOnDateReadable":"December 4th, 2025"},"versionCreatedAt":"2025-09-03 06:39:26","video":"","vorDoi":"10.1186/s12967-025-07529-5","vorDoiUrl":"https://doi.org/10.1186/s12967-025-07529-5","workflowStages":[]},"version":"v1","identity":"rs-7424447","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7424447","identity":"rs-7424447","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}