Single-stranded DNA in the bone microenvironment promotes prostate cancer bone metastasis via the ITGA6-FAK pathway

Abstract

Bone metastases represent a critical phenotype of prostate cancer progression, driven by factors within the bone microenvironment. However, the molecular mechanisms underlying this progression remain poorly understood. In this study, we observed a significant accumulation of single-stranded DNA within the metastatic bone microenvironment of PCa patients. Through cell-SELEX methodology, we identified a PCa target-specific ssDNA, EHBP1. Specifically, EHBP1-ssDNA specifically captures PCa cells by binding to the transmembrane protein integrin α6, which subsequently activates the integrin α6-FAK signaling pathway. Functional studies revealed that knockdown of integrin-α6 expression effectively abrogated EHBP1-ssDNA mediated PCa bone metastatic capacity. Notably, these findings were recapitulated through pharmacological inhibition of FAK signaling using Defactinib, an FAK-specific inhibitor. Taken together, our findings reveal that bone-marrow ssDNA may represent a bone microenvironment factor that captures and promotes PCa homing to bone, further suggesting a potential therapeutic strategy for mitigating bone metastasis.

Bone marrow ssDNA, specifically EHBP1-ssDNA, drives prostate cancer bone metastasis via integrin α6-FAK signaling, and Defactinib blocks this pathway.

Introduction

Prostate cancer (PCa) is the second most commonly diagnosed cancer in men and one of the leading cancer types for the estimated new cancer deaths^1,2. Although the overall incidence of PCa has decreased due to the advent of PET/CT imaging techniques and early detection of metastatic disease, the proportion of patients diagnosed with bone metastatic disease at the time of diagnosis has risen over the past decade^3,4. Clinically, anti-androgen therapies remain the clinical standard of care for advanced PCa. However, despite initial therapeutic responses, a substantial proportion of patients with advanced PCa will ultimately progress to castration-resistant PCa despite continued anti-androgen treatment^5,6. By further elucidating the cellular and molecular mechanisms underlying bone metastasis in PCa, we may facilitate the development of targeted therapies. Such advancements hold significant promise for improving clinical outcomes and enhancing the survival rates of patients with metastatic disease.

Emerging evidence has established that alterations within the primary tumor microenvironment represent a critical determinant driving PCa progression and metastatic colonization. These modifications, originating from genetic aberrations and modulated by non-epithelial stromal components—including cancer-associated fibroblasts, tumor-infiltrating immune cells, and endothelial cells—create a permissive niche that facilitates PCa cell growth, invasion, and dissemination⁷. Certain PCa cells, having acquired migratory capabilities, will enter the blood or lymphatic circulation by decreasing their adhesion to the cells and extracellular matrix (ECM) of the tumor microenvironment, and subsequently home to secondary organs, particularly bone and brain. Prior to progression, the primary tumor remotely triggers a pathological remodeling of bone tissue by releasing extracellular vesicles or signaling molecules (such as interleukins, vascular endothelial growth factors (VEGFs), and TGF-β), thereby establishing a “pre-metastatic niche” in secondary organs^8,9. These have entered circulating malignant tumor cells over-expressing ligands such as integrins β1, α4β1, and α5β1, cadherin-11, connective tissue growth factor, and CXCR4 that interact with receptor proteins present in the bone marrow stroma, including the urokinase receptor, vascular cell adhesion molecule-1, and fibronectin. This interaction facilitates their homing to the bone marrow^10–12. These findings indicate that the biological and molecular remodeling of the bone microenvironment is attributed to the preferential metastasis of tumor cells to bone. Certainly, a deeper understanding of the intricate interactions within the tumor-bone microenvironment and the involvement of multiple signaling pathways in its progression will facilitate the development of potent strategies to inhibit disease progression.

Bone marrow supernatant fluid (BMSF), a specialized biopsy fluid, may function as a reservoir for growth factors that orchestrate tumor bone metastasis, notably TGF-β, bone morphogenetic proteins, insulin-like growth factor, VEGF, and platelet-derived growth factor^13,14. Recent studies have demonstrated the existence of Versican-associated cell-free microRNAs within BMSF, suggesting that this biopsy fluid also harbors nucleic acid fragments¹⁵. So far, however, it remains unclear whether BMSF contains other nucleic acids, such as extracellular DNA (exDNA). According to previous studies, exDNA was indeed detected in various bodily fluids, where it enters circulation following cell death—whether as part of normal physiological cell turnover or pathological processes¹⁶. These exDNA constitute a complex heterogeneous mixture of short DNA fragments, encompassing conventional double-stranded linear segments alongside alternative topological structures such as circular and single-stranded conformations¹⁷. For example, research reports that plasma contains ultra-short single-stranded DNA (ssDNA) in addition to nucleosomal cell-free DNA by ultra-short cell-free DNA sequencing¹⁸. The observation was independently confirmed by another study group¹⁹. Previously researches report that exDNA is relatively short¹⁸, with a length of approximately 50 nucleotides. These short, ssDNA or RNA molecules can be generated through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) that bind to specific molecular targets with high affinity²⁰. It is known that both bone matrix and bone marrow undergo continuous and dynamic turnover processes²¹, strongly suggesting that this process contributes to the formation of exDNA in BMSF. Hence, in this study, we hypothesize that BMSF harbors abnormally generated ssDNA, which may serve to capture circulating PCa cells entering the bone microenvironment and facilitate their adhesion to bone tissues.

In the present study, we discovered that BMSFs derived from PCa patients with bone metastases were enriched in ssDNA. Furthermore, through a cell-SELEX strategy, we identified a high-affinity ssDNA specific to PCa cells, EHBP1-ssDNA. Additionally, we identified ITGA6 as a binding target for EHBP1-ssDNA. The binding of EHBP1-ssDNA to ITGA6 substantially enhances the adhesion and proliferation of PC-3 cells via the integrin α6-FAK pathway, an effect that was reversed upon treatment with Defactinib. Our work presents a theory explaining the preferential skeletal localization of PCa and offers a promising strategy for PCa bone metastasis treatment.

Methods

Clinical samples

Clinical samples were collected from Xiangya Hospital, Central South University, in accordance with the Declaration of Helsinki. The study was approved by the Ethics Committee of Xiangya Hospital (Approval No. 2024081279), and written informed consent was obtained from all participants. All ethical regulations relevant to human research participants were followed.

Serum samples were obtained from 20 treatment-naïve patients with prostate bone metastasis and 20 non-cancer controls (n = 20 per group). Bone marrow supernatants were collected from a subset of 3 patients per group with traumatic fractures (n = 3 per group).

All enrolled patients met the inclusion criteria (metastasis group: histologically confirmed, untreated PCa with bone metastasis; control group: no cancer, undergoing fracture surgery). Randomization was not applicable, as patients were allocated based on disease status. To minimize potential confounders, all samples were processed in a blinded manner and in random order within each batch. No samples were excluded due to quality issues or missing data; thus, all 20 serum samples per group and 3 bone marrow supernatants per subgroup were included in the final analyses. To ensure strict adherence to ethical standards, bone marrow samples from controls were derived exclusively from surgical waste tissue generated during routine procedures; no additional or unnecessary biopsies were performed on healthy individuals.

Mice

Six-week-old male wild-type BALB/c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and bred and maintained by Hunan SJA Laboratory Animal Company (China). All animal work was performed in strict accordance with all relevant ethical regulations for animal use and was approved by the Animal Ethics Committee at Hunan SJA Laboratory Animal Company (IACUC-SJA2023110).

A total of 80 mice were used in this study. Mice were randomly assigned to experimental groups using a random number table. To minimize confounding factors: (i) surgeries and treatments were performed in an alternating order between groups; (ii) bioluminescence imaging (BLI) measurements were conducted at consistent times of day and in randomized cage order; (iii) investigators performing outcome assessments were blinded to group allocation.

All animals completed the study, and no exclusions were made; therefore, data from all 80 mice were included in the final analyses. In each experiment, mice were randomly assigned to experimental groups as described below, with 8 mice per group: Experiment for Fig. 1: Group 1 (vehicle + scrambled DNA, n = 8) and Group 2 (scrambled DNA + ssDNA, n = 8). Experiment for Fig. 3: sgControl group (n = 8), sgITGA6 group (n = 8), sgControl + EHBP1-ssDNA group (n = 8), and sgITGA6 + EHBP1-ssDNA group (n = 8). Experiment for Fig. 5: Vehicle group (n = 8), Defactinib group (n = 8), Vehicle + EHBP1-ssDNA group (n = 8) and Defactinib + EHBP1-ssDNA group (n = 8).

Fig. 1. Bone metastases in prostate cancer are linked with ssDNA.

A Schematic of the workflow for the extraction and analysis of ssDNA from human bone marrow supernatants. B Chromatogram of extracted ssDNA. C Representative image of agarose gel electrophoresis of ssDNA levels from bone fracture and bone metastasis patients. D The ssDNA concentration in bone marrow supernatants was compared between non-cancer bone fracture patients (normal, n = 3) and bone metastasis patients (met, n = 3). Meanwhile, the ssDNA concentration in serum was compared between non-cancer samples (normal, n = 20) and bone metastasis patients (met, n = 20). E Schematic of the experimental injection protocol. Group 1, vehicle injected into the left femur and scrambled DNA into the right femur. Group 2, scrambled DNA was injected into the left femur and ssDNA into the right femur. Each group included 8 mice. F Representative BLI images from mice in each group (n = 8). G Quantification of the BLI signals measured in bone metastases from each group (n = 8). H Kaplan–Meier survival curve of mice from Group 1 and Group 2 (n = 8). I Representative micro-CT 3D reconstructed images from each group. J Quantitative Micro-CT analysis of the trabecular bone microarchitecture of femurs (n = 8). K Representative H&E staining of bone lesions invaded by tumors from each group. Scale bar, 100 μm. L Quantification of relative tumor area of mice in each group (n = 8). *P < 0.05; ***P < 0.001; ****P < 0.0001. Student’s t-test was used for (D, F, H, J). A two-sided log-rank test was used for the survival difference in (K). The error bar represent standard error of the mean. BV/TV trabecular bone volume per tissue volume, Tb.Th trabecular thickness. A was created with BioGDP.com, and E was created with MedPeer (www.medpeer.cn).

Fig. 3. ITGA6 knockdown suppresses bone metastasis in mice.

A Schematic of the injection protocol and representative BLI images from mice in the sgControl and sgITGA6 groups. B Quantification of the BLI signals of bone metastases in sgControl and sgITGA6 groups (n = 8). C Kaplan–Meier survival curve of mice from sgControl and sgITGA6 groups (n = 8). D Representative micro-CT 3D reconstructed images from mice in the sgControl and sgITGA6 groups. E Quantitative micro-CT analysis of the trabecular bone microarchitecture from the femurs of mice in sgControl and sgITGA6 groups (n = 8). F Schematic of injection protocol and representative BLI images from mice in sgControl + EHBP1-ssDNA and sgITGA6 + EHBP1-ssDNA groups. G Quantification of the BLI signals of bone metastases in sgControl + EHBP1-ssDNA and sgITGA6 + EHBP1-ssDNA groups (n = 8). H Kaplan–Meier survival curve of mice from sgControl + EHBP1-ssDNA and sgITGA6 + EHBP1-ssDNA groups (n = 8). I Representative micro-CT 3D reconstructed images from mice in sgControl + EHBP1-ssDNA and sgITGA6 + EHBP1-ssDNA groups. J Quantitative micro-CT analysis of the trabecular bone microarchitecture from the femurs of mice in sgControl and sgITGA6 groups (n = 8). K Representative H&E images and quantification of bone lesions invaded by tumors from sgControl and sgITGA6 groups (n = 8). Scale bar, 100 μm. L Representative H&E images and quantification of bone lesions invaded by tumors from sgControl + EHBP1-ssDNA and sgITGA6 + EHBP1-ssDNA groups (n = 8). Scale bar, 100 μm. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Student’s t-test was used for (B, E, G, J, K, L). A two-sided log-rank test was used for survival difference in (C, H). The error bar represent standard error of the mean. BV/TV trabecular bone volume per tissue volume, Tb.Th trabecular thickness. A, F were created with MedPeer (www.medpeer.cn).

Fig. 5. Defactinib functions as a FAK inhibitor and blocks bone metastasis in vivo.

A Representative BLI images from mice in the Vehicle and Defactinib groups. B Kaplan–Meier survival curve of mice in Vehicle, Defactinib, Vehicle + EHBP1-ssDNA, and Defactinib + EHBP1-ssDNA groups (n = 8). C Representative BLI images from mice in Vehicle + EHBP1-ssDNA and Defactinib + EHBP1-ssDNA groups. D Quantification of the BLI signals of bone metastases in each group (n = 8). E Representative micro-CT 3D reconstructed images from mice in Vehicle and Defactinib groups. F Quantitative micro-CT analysis of the trabecular bone microarchitecture of mice femurs from Vehicle and Defactinib groups (n = 8). G Representative micro-CT 3D reconstructed images from mice in Vehicle + EHBP1-ssDNA and Defactinib + EHBP1-ssDNA groups. H Quantitative micro-CT analysis of the trabecular bone microarchitecture of mice femurs from Vehicle + EHBP1-ssDNA and Defactiib + EHBP1-ssDNA groups (n = 8). I Representative H&E images and quantification of relative tumor area from mice in Vehicle and Defactinib groups (n = 8). Scale bars represent 100 μm. J Representative H&E images and quantification of relative tumor area from mice in Vehicle + EHBP1-ssDNA and Defactinib + EHBP1-ssDNA groups (n = 8). Scale bars represent 100 μm. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Student’s t-test was used for (D, F, H, I, J). A two-sided log-rank test was used for the survival difference in (B). The error bar represent standard error of the mean. BV/TV trabecular bone volume per tissue volume, Tb.Th trabecular thickness.

Sample size (n = 8 per group) was determined a priori by power analysis based on pilot data (primary outcome: BLI signal), which indicated that 7 mice per group were sufficient to achieve 80% power at α = 0.05. We increased this to 8 to account for potential technical variability, while adhering to the 3Rs principles. We have complied with all relevant ethical regulations for animal use.

Cell culture

Human PCa cell line C4-2B (ZQ1110) and Luciferase-expressing PCa cell line (PC-3-luc, LZQ0024) were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. The human breast cancer cell lines ZR-75-1 (CL-0247), MDA-MB-231 (CL-0150), and MCF-7 (CL-0149), as well as the non-tumorigenic human prostate epithelial cell line RWPE-1 (catalog no. CL-0200), were purchased from Wuhan Pricella Biotechnology Co., Ltd. C4-2B, PC-3-luc, and RWPE-1 were derived from male donors; ZR-75-1, MDA-MB-231, and MCF-7 from female donors. None of these cell lines appears in the ICLAC Register of Misidentified Cell Lines (version 13, http://iclac.org/databases/cross-contaminations).

Cell lines were not re-authenticated by the authors but were certified mycoplasma-negative by suppliers prior to distribution using standardized PCR-based methods. C4-2B and PC-3-luc cells were cultured in Ham’s F12 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich) and 1% penicillin/streptomycin (P/S; Solarbio). ZR-75-1 cells were maintained in RPMI-1640 medium (Gibco) supplemented with 10% FBS (Sigma-Aldrich) and 1% P/S (Solarbio). MDA-MB-231 and MCF-7 cells were grown in DMEM (Gibco) containing 10% FBS and 1% P/S. RWPE-1 cells were cultured in the specific culture medium for RWPE-1 cells (Pricella, CM-0200). All cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂.

Tumor implantation and bioluminescence analysis

For the intracardiac injection model, 1 × 10⁶ PC-3-luc cells were injected into the left cardiac ventricle of anesthetized mice. The maximum tumor burden permitted by the ethics committee was defined as: (1) primary tumor volume not exceeding 1800 mm³; (2) metastatic burden causing less than 18% body weight loss; and (3) absence of ulceration, necrosis, or signs of distress. Throughout the experimental period, all animals were monitored daily for signs of distress, body weight changes, and overall health condition. The maximal tumor size/burden permitted by the ethics committee was not exceeded in any animal in this study. Animals were euthanized immediately if they exhibited signs of severe distress, rapid weight loss (>18% of initial body weight), neurological deficits, or hind limb paralysis.

The development of metastases was monitored by BLI. In vivo bioluminescence imaging was performed weekly with IVIS Spectrum (PerkinElmer). Anesthetized mice were intraperitoneally injected with 100 μl of 15 mg/kg D-Luciferin (Invitrogen, L2912). Analysis was performed with Living Image software (PerkinElmer), and all images were normalized to the same radiance scale. Average radiance intensity was quantified in units of radiance (p s⁻¹ cm⁻² sr⁻¹). In order to quantify luminescence in specific areas of the mice, regions of interest were drawn around the right leg.

Adhesion, migration, and invasion assays

The adhesion assay was performed using 96-multi well plates coated with 10 μg/ml fibronectin (Solarbio, F8180). Cells were seeded inside wells containing culture media without serum. After 24 h, cells that adhered to the bottom of the wells were fixed in 4% formaldehyde. The fixed cells were stained using 0.1% crystal violet (Solarbio, G1063). The assay was quantified by counting the stained nuclei in five independent fields of five repeated wells.

Assays of migration and invasion were performed using 6.5-mm transwell inserts (Corning, 3422) coated with nothing or growth-factor-reduced Matrigel (Corning, 354230), respectively. Cells were serum-starved overnight before seeding to eliminate the interference of proliferative effect with cell migration or invasion. Cells were seeded inside transwell inserts containing culture media without serum. After 16 h, cells that translocated to the lower surface of the filters were fixed in 4% formaldehyde. The fixed membranes were stained using 0.1% crystal violet. Assays were quantified by counting the stained nuclei in five independent fields of five repeated transwells.

Colony formation assay

To evaluate the proliferation capability, a colony formation assay was performed. Complete medium mentioned in the cell culture method section above, containing 10 nM of candidate ssDNA, Scrambled DNA, or maintained under conditions for ITGA6 knockdown or not (transfection with sgRNA), was applied to PC-3 cells. Subsequently, the pre-treated cells were harvested and seeded into 12-well plates at a density of 200 cells per well. The cells were cultured in complete medium containing the respective treatments at 37 °C for 10 days to allow colony formation. The culture medium was refreshed every 3 days. After incubation, the colonies were washed with PBS, fixed with 4% paraformaldehyde for 20 min, and stained with 0.1% crystal violet solution for 10 min.

Hydroxyapatite chromatography

Human bone marrows were collected and subjected to centrifugation to separate the BMSF. BMSF was injected into a clean centrifuge tube, filtered through a 100-um cell filter, and centrifuged at 1200 r/min for 5 min. The supernatant was collected. The DNA from the supernatant was then extracted through treatment with the MagMAX DNA Multi-Sample Ultra 2.0 Kit (Thermo Scientific, A36570). Combine DNA with 0.12 M phosphate buffer in a final volume of 500 μl and incubate at 60 °C for 10 min. Allow the DNA to bind to the Illustra NAP-5 Columns (Cytiva, 17085301) for 30 min. Open the stopcock and collect the initial ssDNA sample in the 15 ml tube. Add 6 ml of 0.12 M phosphate buffer to the columns, and collect ssDNA in the same tube. Add 1 volume of phenol: chloroform: isoamyl alcohol (25:25:1 v/v/v) solution to the tube, mix vigorously, and centrifuge at 3500 × g for 15 min. Transfer supernatant containing ssDNA to a new 15 ml tube. Desalt collected ssDNA samples with an Amicon Ultra-4 centrifugal filter device outfitted with a 30,000 molecular weight cut-off Ultracel membrane (Merck, UFC803024). Schematics were created with BioGDP.com²² and MedPeer (www.medpeer.cn).

Cell-SELEX selection

The initial DNA library was generated from ssDNA isolated from BMSF of PCa patients with bone metastasis, following the protocol (ABclonal RK20222) and dissolved in binding buffer (0.01 M pH 7.4 PBS containing 5 mM MgCl₂, 0.2 mg/mL tRNA, 1 mg/mL salmon sperm DNA, 2 mg/mL BSA)^23,24. After denaturation at 95 °C for 5 min and immediate cooling on ice for 10 min, the initial library was incubated for 2 h with PC-3 cells cultured at 90% confluence in 100 mm dishes at 4 °C. After incubation, the supernatant was removed, and the cells were washed with washing buffer. PC-3 cells were then harvested and transferred to 500 μL of water and heated for 10 min at 95 °C to collect cell-bound DNA. FAM-labeled forward primers and biotin-labeled reverse primers were prepared by PCR using cell-bound DNA as a template (6–14 cycles of 30 s at 95 °C, 30 s at 58 °C, 30 s at 72 °C, and then 30 s at 72 °C for 5 min). The double-stranded DNA (dsDNA) product was then separated from the PCR solution by streptavidin-coated agarose beads (GE Healthcare). After treatment with 0.2 M NaOH, FAM-labeled ssDNA was separated from dsDNA, desalted, and lyophilized for the next round of selection. Starting from the third round, the evolved ssDNA pool was first incubated with RWPE-1 cells in 60 mm Petri dishes at 90% confluence for 30 min at 4 °C for subtractive selection. Unbound ssDNA was then removed and applied to PC-3 cells. To increase the stringency of selection, the positive incubation time was shortened from 2 to 1 h, and the washing time was gradually extended from two to three times as the number of selection rounds increased. Meanwhile, the negative incubation time was gradually increased from 1 to 2 h. Starting from the 9th round of selection, two consecutive subtractive selections were carried out to maximize the removal of ssDNA bound to normal prostate cells. The ssDNA library derived from the 12th round of iterative selection underwent PCR amplification, followed by cloning into a T-vector system and subsequent Sanger sequencing (services provided by Sangon Biotech Co., Ltd.). The names of ssDNAs were based on their respective gene names. The “EHBP1-ssDNA,” for instance, is so named because its sequence maps specifically to the EHBP1 gene.

Flow Cytometry Analysis

To evaluate the binding ability of candidate ssDNA to cells, flow cytometry was performed. In brief, PC-3 cells (2 × 10⁵ cells) were incubated with 250 nM candidate FAM-labeled ssDNA or FAM-labeled libraries in 250 μL of binding buffer for 1 h at 4 °C. The cells were then washed three times with washing buffer and resuspended in 200 μL of D-PBS for analysis.

To determine the dissociation constant (Kd), a saturation binding assay was conducted, and PC-3 cells (3 × 10⁵) were incubated with different concentrations (0, 20, 50, 100, and 200 nM) of ssDNA in 250 μL of binding buffer for 1 h at 4 °C. After incubation, each sample was washed three times with washing buffer and then resuspended in 200 μL D-PBS for flow cytometry analysis. These experiments were repeated three times. Using GraphPad Prism 9.5 software, the Kd of ssDNA was calculated by Equation Y as Bmax × X/(Kd + X) (Y: relative fluorescence intensity; X: ssDNA concentration), fitting the dependence of the fluorescence intensity of the cell/ssDNA complexes on the ssDNA concentration.

Micro-CT analysis and histochemistry of the mouse femur

The fresh femurs were dissected and fixed in 4% paraformaldehyde for 24 h. The fixed samples were scanned using high-resolution micro-CT (Skyscan 1172, Bruker micro-CT) following a previously described method²⁵. After micro-CT analysis, the femurs were decalcified in 10% EDTA for 2 weeks, embedded in paraffin, and cut to 4 µm. As described previously, the 4-µm paraffin sections were stained with hematoxylin-eosin²⁶.

Cyto-immunofluorescent staining

PC-3 cells were cultured for 24 h and fixed in 4% paraformaldehyde for 20 min. Then, cells were permeabilized with 0.25% Triton X-100 (Sigma-Aldrich, T8787) for 20 min, and incubated with primary antibodies against ITGA6 (R&D Systems, MAB13501, 1:100) and ITGB4 (Santa Cruz Biotechnology, sc-514426, 1:50), followed by incubation with secondary antibodies. Nuclei were counterstained with DAPI (Beyotime, C1006). The fluorescent signals were captured via fluorescence microscopy (Apotome 3, Zeiss).

Western blotting

Western blotting was performed as described previously²⁷. Blots were incubated with primary antibodies described as the following: FAK (Forevertech Biotechnologies Co., Ltd, 20431, 1:1000), p-FAK (Tyr397) (CST, 3283S, 1:1000), AKT (CST, 9272S, 1:1000), p-AKT (CST, 4060S, 1:1000), ERK (CTS, 9102S, 1:1000), p-ERK (CST, 4370S, 1:1000), ITGA6 (proteintech, 27189-1-AP, 1:1000), ITGB4 (proteintech, 21738-1-AP, 1:1000), and GAPDH (Origene, TA802519, 1:5000). Secondary antibodies including Goat anti-Mouse IgG (H + L) Secondary Antibody, HRP (Invitrogen, AB_228307) and Goat anti-Rabbit IgG (H + L) Secondary Antibody, HRP (Invitrogen, AB-228341) were subsequently applied.

Immunoprecipitation (IP)

Briefly, PC-3 cells were lysed in the cell lysis buffer (Beyotime, P0013D) with protease inhibitor (Selleck, B14001) for 10 min on ice, and then centrifuged at 13,000 × g for 5 min. The supernatants were collected and incubated overnight at 4 °C with ~5 µg ITGA6 or IgG antibody and Protein A/G magnetic beads (MCE, HY-K0202). After washing four times with IP buffer, Magnetic Beads were boiled with 2× SDS loading buffer, followed by Western blotting using ITGA6 (Proteintech, 27189-1-AP, 1:1000) and FAK (Forevertech Biotechnologies Co., Ltd, 20431, 1:1000).

Biotin pull-down assay

We got ssDNA labeled with biotin from Sangon Biotech (Shanghai) Co., Ltd. PC-3 cells were lysed in the cell lysis buffer (Beyotime, P0013D) with protease inhibitor (Selleck, B14001) for 10 min on ice, and then centrifuged at 13,000 × g for 5 min. To perform the binding assay, biotin-labeled ssDNA was incubated with the supernatant and Protein A/G magnetic beads (MCE, HY-K0202) overnight at 4 °C. Non-labeled ssDNA was used as a negative control. After washing four times with IP buffer, magnetic beads were boiled with 2× SDS loading buffer, followed by Western blotting using ITGA6 (proteintech, 27189-1-AP, 1:1000) and ITGB4 (proteintech, 21738-1-AP, 1:1000).

ssDNA treatment

Scrambled ssDNA sequence with a length of 80 bp and EHBP1-ssDNA were synthesized by Sangon Biotech (Shanghai) Co., Ltd. For the injection of either a scrambled ssDNA sequence or EHBP1-ssDNA, intra-bone marrow delivery was performed following a previously described method²⁸. The scrambled ssDNA and EHBP1-ssDNA were diluted with sterile 1×PBS to a final concentration of 50 nM. Fifty microliters of each solution were injected into the bone marrow cavity of the right femur twice weekly for 1 month.

ssDNA binding assay

PC-3 cells (1 × 10⁴ cells), including a control cell line and an ITGA6-knockdown cell line, were seeded into 24-well cell culture plates and cultured in F12 medium supplemented with 10% FBS and 1% P/S. One hour prior to the assay, the culture medium was replaced with fresh medium containing 1 mg/mL salmon sperm DNA and 0.2 mg/mL tRNA, and the cells were incubated at 37 °C for 1 h. EHBP1-ssDNA sequences with FAM modification and a control scrambled sequence with a length of 80 bp were denatured at 70 °C for 3 min and then allowed to refold at room temperature for 20 min. These refolded ssDNA sequences were added to the seeded cells in full growth medium and incubated at 37 °C for 1 h unless otherwise specified. One hour later, the cell supernatant was removed, and the cells were washed three times with 1×PBS. Images were captured using an inverted fluorescent microscope (Ti2-A, Nikon).

Mass spectrometry analysis

For mass spectrometry analysis, the protein isolated via both biotin pull-down and His-tag pull-down assays was subjected to enzymatic digestion, desalting, concentration, and final reconstitution. Each sample was labeled using TMT reagent (Thermo Scientific). LC-MS/MS analysis was performed as described²⁹.

RNA isolation and qRT-PCR analysis

RNA isolation and qRT-PCR analysis were performed as described previously³⁰. The primer sequences were listed as follows: GAPDH: Forward-5′-GGAGCGAGATCCCTCCAAAAT-3′, Reverse-5′-GGCTGTTGTCATACTTCTCATGG-3′; ITGA6 Forward-5′-TGGCGTGGCTGACTT ACAT-3′, Reverse-5′-TGGCGTGGCTGACTTACAT-3′; ITGB4: Forward-5′-CCAGGAAGAUCC AUUUCAATT-3′, Reverse-5′-UUGAAAUGGAUCUUCCUGGTT-3′.

Defactinib treatment

Defactinib (MedChemExpress, 1073154-85-4) at 25 mg/kg was administered via oral gavage once a day for 4 weeks. Control mice received vehicle only. The EHBP1-ssDNA was injected into the bone marrow cavity of the right femur twice a month for 1 month.

Statistics and reproducibility

Statistical analyses were performed with GraphPad Prism 9.5. The data are presented as mean ± SEM. Comparisons between two experimental groups were assessed using unpaired two-tailed Student’s t-tests. For comparisons involving three or more groups, one-way analysis of variance (ANOVA) with Tukey’s post hoc test was applied. Survival differences were evaluated using the two-sided log-rank test. P < 0.05 was used to define statistical significance and indicated by “*”; P < 0.05 was indicated by “**”; P < 0.01 was indicated by “***”; P < 0.001 was indicated by “****.”

Sample sizes were determined based on power calculations from pilot studies and established precedents in comparable pre-clinical models. In vivo experiments utilized eight mice per experimental group (n = 8), representing biological replicates. Human sample analyses included bone marrow supernatants from three non-cancer bone fracture patients (control) and bone metastasis patients (sample sizes detailed in figure legends), and serum samples from twenty non-cancer individuals and bone metastasis patients. For in vitro studies, experiments were independently repeated at least three times (as specified: n = 3 or n = 5 independent experiments), with each independent experiment constituting a separate biological replicate performed on different days. Technical replicates (e.g., triplicate wells in binding or functional assays) were averaged for each independent experiment. All representative images and quantitative data presented are derived from these independent replicates, confirming the reproducibility of the results.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Results

ssDNA associates with bone metastases in prostate cancer

To determine whether BMSF contains ssDNA, we collected bone marrow samples from three PCa patients with bone metastasis and concomitant bone fracture, as well as three normal bone marrow samples from Xiangya Hospital. Total nucleic acid was extracted from BMSF and then loaded onto hydroxyapatite chromatography for the isolation of ssDNA (Fig. 1A). We successfully isolated ssDNA from bone marrow samples and observed that it has an length of about 80 bps (Fig. 1B). Furthermore, the ssDNA concentration in the bone microenvironment and serum was significantly higher in patients with bone metastases (met) compared to those non-cancer fracture samples (normal), as quantified by gel electrophoresis and concentration assays (Fig. 1C, D). The results suggest an association between elevated ssDNA levels and the presence of bone metastasis, potentially reflecting increased cell turnover/damage in advanced cancers. Additionally, it raises the possibility that ssDNA may contribute to the remodeling of the bone microenvironment. Thus, we put forward this finding as a hypothesis-generating observation, warranting further mechanistic investigation.

To further investigate whether BMSF ssDNA is involved in the progression of bone metastasis in PCa, we inoculated PC-3-luc cells into the left ventricles of nude mice to establish systemic bone metastases. Subsequently, we locally injected BMSF ssDNA into the femurs as follows: in Group 1, vehicle was delivered to the left femur and scrambled DNA into the right femur; in Group 2, scrambled DNA was injected into the left femur and ssDNA into the right femur (Fig. 1E). Bioluminescence images were captured weekly, and images at 4th week were used for analysis. We found that there was no difference between femurs treated with scrambled DNA and vehicle, while there was significantly greater tumor area burden in femurs injected with ssDNA compared to scrambled DNA (Fig. 1F, G). Survival analysis revealed significantly reduced longevity in group 2 compared to group 1 (Fig. 1H). Post-euthanasia analysis demonstrated concordant results between imaging and histological assessments: ssDNA-injected bones exhibited marked bone deterioration through micro-CT quantification, showing reduced trabecular bone volume (BV/TV) and decreased trabecular thickness (Tb.Th) (Fig. 1I, J) and displayed larger tumor volumes by H&E staining (Fig. 1K, L). These findings demonstrate that ssDNA, derived from the BMSF of PCa patients, possesses the ability to enhance bone metastasis in PCa.

EHBP1-ssDNA promotes prostate cancer bone metastasis by binding to integrin α6

Based on our findings, the ssDNA isolated from the BMSF of PCa patients is approximately 80 nucleotides in length, which we hypothesized to exhibit high affinity specifically for PCa. Using PC-3 cells, a bone metastasis-derived standard model for PCa studies^31,32, as targets and normal RWPE-1 prostate cells for counter-selection, we conducted 12 rounds of cell-SELEX with patient-derived ssDNA directly as the initial library (Fig. 2A). Deep sequencing identified three predominant ssDNA by Round 12, among which ssDNA EHBP1 exhibited superior binding affinity to PC-3 cells as demonstrated by flow cytometry (Fig. 2B, C). Quantitative affinity assessment using FAM-labeled EHBP1-ssDNA showed concentration-dependent binding (Fig. 2D) to PC-3 cells, a human PCa cell line (Fig. 2E), indicating the high-affinity interaction of EHBP1-ssDNA with metastatic PCa cells (Fig. 2D). Short, ssDNA or RNA oligonucleotides, such as aptamers, can bind to target molecules with high specificity and affinity^6,24,33. By modulating the downstream signaling pathways of their target proteins, these oligonucleotides trigger cascade reactions at both cellular and molecular levels, ultimately inducing functional alterations in biological systems³⁴. To identify the target molecule for EHBP1-ssDNA, we synthesized biotin-labeled EHBP1-ssDNA and incubated it with membrane proteins from PC-3, C4-2B (PCa), and MDA-MB-231 cells (a highly metastatic triple-negative breast cancer line)³⁵ as malignant controls, contrasting with RWPE-1 (non-tumorigenic prostate) cells and ZR-75-1 (non-metastatic ER⁺ breast cancer)^36,37 as isogenic controls. Comparative membrane proteome analysis identified proteins pulled down by biotin-modified EHBP1-ssDNA, among which ITGA6 and PLP2 exhibited high affinity and specific expression in bone metastatic cell lines (PC-3, C4-2B and MDA-MB-231) compared to non-metastatic cell lines (RWPE-1 and ZR-75-1) (Fig. 2F, G). Among them, ITGA6, a metastasis-associated adhesion receptor, is recognized for its heterodimerization with ITGB4 to form the integrin α6β4 complex, which regulates kinase signaling pathways in PCa^38,39.

Fig. 2. EHBP1-ssDNA contributes to the bone metastasis of prostate cancer and interacts with integrin α6.

A Schematic of the cell-SELEX protocol. B Abundance of the top 7 sequences from cell-SELEX cycle 12. C The emission spectrum of FAM-conjugated candidate ssDNA binders. FAM-ssDNA was incubated with PC-3 cells at 37 °C for 30 min at a concentration of 200 nM. D The emission spectrum of FAM-conjugated EHBP1-ssDNA at different concentrations. FAM-EHBP1-ssDNA was incubated with PC-3 cells at 37 °C for 30 min at a series of concentrations. E Determination of the equilibrium dissociation constant via concentration-dependent binding of EHBP1-ssDNA to PC-3 cells (0–200 nM). Kd was calculated via the GraphPad Prism program 9.5 (n = 3 for each concentration). F Venn diagram illustrating the overlap of membrane proteins identified by biotin-EHBP1-ssDNA pull-down assays in C4-2B, PC-3, MDA-MB-231, RWPE-1, and ZR-75-1 cell lines. G Mass spectrogram of the ITGA6 protein tandem mass spectrum. H Representative images of co-localization of ITGA6 (green), Dil (red), and DPAI (blue) immunostaining in PC-3 cells. Scale bar, 25 μm. I RT-qPCR analysis of ITGA6 and ITGB4 expression levels in PC-3, MDA-MB-231, RWPE-1, and ZR-75-1 cells. n = 3 independent experiments. J Western blot analysis of the interaction between biotin-EHBP1-ssDNA and ITGA6/ITGB4. K Representative fluorescence images of EHBP1-ssDNA-FAM or FAM-labeled scrambled DNA sequence binding to sgControl and ITGA6-knockdown PC-3 cell lines. Corresponding bright-field images confirm cell presence. Scale bar, 50 μm. L Quantitative analysis of FAM fluorescence in sgControl and ITGA6-knockdown PC-3 cell lines treated with EHBP1-ssDNA-FAM or FAM-labeled scrambled DNA (n = 5). M Representative images of cell adhesion and colony formation. Scale bar, 100 μm. N Quantitative analysis of cell adhesion and colony formation. n = 5 independent experiments. n.s. not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by one-way ANOVA with Tukey’s post hoc test for multiple comparisons. The error bar represent standard error of the mean. A was created with MedPeer (www.medpeer.cn).

Therefore, following subsequent research, we focused on the EHBP1-ssDNA target molecule, ITGA6. Using DiI, a cell membrane dye, we performed fluorescence co-localization studies, which showed that ITGA6 colocalizes with DiI staining, indicating that ITGA6 was specifically expressed in the cell membrane of PC-3 cells (Fig. 2H). RT-qPCR analysis demonstrated that the expression levels of ITGA6 were highest in PC-3 cells when compared to other cell lines (Fig. 2I). Biotin-conjugated EHBP1-ssDNA pull-down assays with magnetic beads followed by immunoblotting demonstrated direct physical interaction between EHBP1-ssDNA and both ITGA6/ITGB4 subunits (Fig. 2J). Additionally, genetic silencing of ITGA6 using sgRNA in PC-3 cells significantly attenuated EHBP1-ssDNA binding (Fig. 2K) and reduced the adhesion capacity induced by EHBP1-ssDNA treatment (Fig. 2L, M). These findings indicate that EHBP1-ssDNA not only effectively captures PCa cells but also promotes their proliferative activity, confirming integrin α6 as its functional receptor.

EHBP1-ssDNA facilitates PCa bone metastasis via integrin α6

To examine whether EHBP1-ssDNA facilitates PCa bone metastasis through ITGA6 modulation in vivo, PC-3-luc cells were transduced with sgITGA6 or sgControl lentiviral particles to establish stable ITGA6-knockdown cell lines utilizing the CRISPR-Cas9 genome editing system. The stable ITGA6-knockdown and control cell lines were inoculated into the left ventricles of nude mice. Bioluminescence images were captured weekly, and images at 4th week were used for analysis. Compared to vehicle-treated controls, the sgITGA6 group presented diminished bone metastatic foci (Fig. 3A, B), improved survival outcomes (Fig. 3C), and enhanced trabecular bone preservation as demonstrated by micro-CT analysis (Fig. 3D, E).

We repeated left ventricles injection of ITGA6-knockdown and control PC-3-luc cells, and further injected EHBP1-ssDNA into the bone marrow cavity of the right leg in mice, to determine whether ITGA6 is required for EHBP1-ssDNA function. Similarly, the knockdown of ITGA6 was observed to modestly diminish EHBP1-ssDNA’ s pro-metastatic activity in the sgITGA6 + EHBP1-ssDNA treatment group, indicated by bioluminescence (Fig. 3F, G), survival analysis (Fig. 3H), micro-CT (Fig. 3I, J), and H&E staining (Fig. 3K, L), as compared to the sgControl+EHBP1-ssDNA group. Collectively, these findings demonstrate that EHBP1-ssDNA facilitates PCa metastasis through interaction with the ITGA6 receptor, whereas ITGA6 knockdown partially counteracts EHBP1-ssDNA’ s pro-metastatic activity.

EHBP1-ssDNA activates the integrin α6-FAK signaling pathway in PCa

In order to identify the downstream proteins involved in the oncogenic pathways mediated by EHBP1-ssDNA/ITGA6, we performed His-tagged ITGA6 pull-down assays using total membrane proteins from PC-3 cells, followed by LC-MS/MS proteomic profiling. This approach robustly identified focal adhesion kinase (FAK) as a high-confidence interacting partner of ITGA6 (Fig. 4A, B). Critically, co-immunoprecipitation experiments confirmed a direct physical interaction between endogenous ITGA6 and FAK in PC-3 cells (Fig. 4C), establishing a molecular link between this integrin and a master regulator of cancer progression. It is well established that the activation of FAK/AKT and FAK/ERK signaling pathways is vital for cancer progression^40,41. Notably, EHBP1-ssDNA treatment induced robust, dose- and time-dependent phosphorylation of FAK at Tyr397, an activation marker in PC-3 cells⁴², concomitant with downstream AKT and ERK activation (Fig. 4D–G), key effectors in tumor survival and migration.

Fig. 4. Integrin α6β4-FAK signaling pathway is activated by EHBP1-ssDNA in PCa.

A Schematic of the ITGA6 pull-down assay and LC-MS/MS analysis. B Mass spectrogram of the FAK protein tandem mass spectrum. C Co-immunoprecipitation (co-IP) analysis of the interaction between FAK and ITGA6 with or without EHBP1-ssDNA treatment performed using an antibody to pull-down extracted proteins from PC-3 cells. Western blotting analysis (D) of ITGA6, ITGB4, p-FAK(Try397), p-AKT, p-ERK, FAK, AKT, and ERK protein levels in PC-3 cells treated with different concentrations of EHBP1-ssDNA for 4 h, and quantification from three independent experiments (E). GAPDH was used as a loading control. Western blotting analysis (F) of ITGA6, ITGB4, p-FAK(Try397), p-AKT, p-ERK, FAK, AKT, and ERK protein levels in PC-3 cells treated with 10 nM EHBP1-ssDNA for different times, and quantification from three independent experiments (G). GAPDH was used as a loading control. Western blotting analysis (H) of p-FAK(Try397), p-AKT, p-ERK, FAK, AKT, and ERK protein levels in sgControl or sgITGA6-transfected PC-3 cells treated with scrambled DNA or EHBP1-ssDNA or blank control, and quantification from three independent experiments (I). GAPDH was used as a loading control. Western blotting analysis (J) of p-FAK(Y397), p-AKT, p-ERK, FAK, AKT, and ERK protein levels in PC-3 cells treated with or without EHBP1-ssDNA or Defactinib, and quantification from three independent experiments (K). GAPDH was used as a loading control. L Representative images of cell adhesion (top), invasion (middle), and migration (bottom) from scrambled DNA, EHBP1-ssDNA, Defactinib + scrambled DNA, Defactinib + EHBP1-ssDNA groups. Scale bar, 100 μm. M Quantitative analysis of cell adhesion, invasion, and migration from each group. n = 5 independent experiments.*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by one-way ANOVA with Tukey’s post hoc test. The error bar represent standard error of the mean.

To determine whether EHBP1-ssDNA activates the FAK pathway through ITGA6-mediated mechanisms, we cultured both ITGA6-knockdown and control PC-3-luc cells, treated them with EHBP1-ssDNA or scrambled DNA, and then analyzed FAK/AKT/ERK activation by western blotting. Strikingly, genetic knockdown of ITGA6 completely abolished EHBP1-ssDNA-induced phosphorylation of FAK, AKT, and ERK (Fig. 4H, I), demonstrating that ITGA6 is essential for transducing EHBP1-ssDNA signals into FAK pathway activation. To further validate our findings, Defactinib, a FAK-specific inhibitor targeting Tyr397 phosphorylation⁴³, was employed to pharmacologically inhibit FAK signaling. Consistent with the results observed in ITGA6-knockdown cells, Defactinib treatment similarly abrogated EHBP1-ssDNA-induced FAK/AKT/ERK activation (Fig. 4J, K), and significantly attenuated EHBP1-ssDNA-enhanced cell adhesion, invasion, and migration in vitro (Fig. 4L, M). Together, we suggested that EHBP1-ssDNA drives metastatic signaling through the integrin α6β4-FAK axis, with FAK phosphorylation serving as a critical mechanistic hub for downstream oncogenic pathway activation.

FAK inhibitor Defactinib attenuates the EHBP1-ssDNA’ s pro-bone metastatic capacity

Notably, the results showed that Defactinib not only suppressed baseline FAK/AKT and FAK/ERK signaling but also reversed EHBP1-ssDNA-induced FAK activation and pro-metastatic effects (Fig. 4J, K), highlighting FAK’s therapeutic potential and prompting in vivo validation. For in vivo validation, PC-3-luc cells were inoculated into the left ventricles of nude mice. Following this, the mice were treated with Defactinib or a control vehicle. Bioluminescence revealed that Defactinib significantly reduced the bone metastasis signals (Fig. 5A) and was associated with improved survival outcomes (Fig. 5B). To further evaluate the therapeutic effect, we repeated the experiment in a parallel cohort of mice in which EHBP1-ssDNA was injected into the right femur. Consistently, Defactinib treatment modestly attenuated the pro-metastatic effect of EHBP1-ssDNA, as evidenced by reduced bioluminescence signals (Fig. 5C, D).

Micro-CT analysis demonstrated that Defactinib preserved bone architecture in both experimental settings, reflected by increased bone volume and trabecular thickness (Fig. 5E–H). Histological examination by H&E staining corroborated these findings, showing smaller tumor volumes in Defactinib-treated mice compared to vehicle controls (Fig. 5I, J). These findings suggested that Defactinib can attenuate the pro-bone metastatic effects of EHBP1-ssDNA by inhibiting FAK.

Discussion

Bone metastases are virtually incurable and result in significant morbidity prior to PCa patients’ death^44,45. Successful colonization of bone metastatic cells requires reciprocal communications with microenvironmental cells and local cell factors. In this study, we propose a theory to explain the propensity of PCa for bone metastasis. Specifically, we demonstrate that ssDNA present in BMSF exhibits properties capable of: (1) capturing circulating PCa cells as they enter the bone microenvironment, and (2) mediating their adhesion to bone tissues.

Some oligonucleotides can interact with the surface structure of target proteins through specific secondary or tertiary structures, offering potential for modulating protein function and conformational changes⁴⁶. While oligonucleotides such as aptamers have been found to exhibit anti-tumor effects in a number of cancer types, including prostate, breast, kidney, and lung cancers^47–49, few studies have mentioned their pro-tumor effects. Here, we demonstrate that BMSF ssDNA exhibits properties akin to neutrophil extracellular traps (NETs), which capture PCa cells infiltrating the bone microenvironment and promote their metastatic colonization in bone. In recent years, treatment based on DNase I, a secreted enzyme capable of degrading NET-DNA, has been applied to mitigate cancer metastasis^50,51. Consistent with these studies, our findings suggest that targeting BMSF-derived ssDNA could represent a feasible therapeutic target to mitigate PCa bone metastasis. We showed that elevated ssDNA concentration in bone marrow supernatant strongly correlates with bone metastasis and provides a pool from which tumor-cell-binding aptamers can be enriched. Additionally, it is worth noting that ultra-short cell-free circulating DNA fragments (~50 nucleotides) in plasma exist predominantly as a single-stranded conformation, as demonstrated by ultra-short cell-free DNA sequencing^17,18. Notably, plasma from cancer patients contains significant amounts of cell-free circulating tumor DNA, which has emerged as a promising liquid biopsy-based biomarker for noninvasive diagnosis, prognosis, and longitudinal monitoring of tumor dynamics across diverse malignancies^52,53. This phenomenon strongly suggests that ssDNA in cancer patient plasma may play a critical role in facilitating the distant metastasis of circulating tumor cells. This will be further confirmed in our subsequent research.

The formation of ssDNA is a common feature of DNA metabolism, occurring during DNA replication, repair, and recombination⁵⁴. Notably, a specific R-loop structure frequently occurs in the process, and it consists of an RNA/DNA heteroduplex along with a looped-out non-template strand. Recent studies have indeed shown that R loops are involved in many human illnesses including cancer, neurological disorders, and autoimmune diseases⁵⁵. In our study, BMSF ssDNA and R-loop structures were mainly located at bone type-H vessels. This observation readily suggests that BMSF ssDNA may originate from the bone type-H vessels. The next question is whether and how PCa cells actively promote bone type-H vessels R-loop formation. Emerging evidence suggests that the dormant and proliferating circulating tumor cells (CTCs) are found in E-selectin and stromal cell-derived factor 1-rich perisinusoidal vascular regions¹¹. Based on these observations, we hypothesize that CTCs interact with bone type-H vessels and trigger R-loop formation through undefined mechanisms. Elucidating these underlying regulatory pathways and the release of BMSF ssDNA will be pivotal in deciphering the intricate cross-talk between CTCs and the bone microenvironment in the future.

Integrins, a class of heterodimeric transmembrane glycoproteins, mediate cell–cell and cell–ECM adhesion while regulating cellular migration. They are widely expressed across both tumor cells and the supporting host stromal cells in the bone microenvironment. Through interactions with ECM components, integrins enable tumor cells to evade cell–cell and cell–matrix constraints, thereby facilitating invasion, migration, and metastatic colonization within new tissues and matrices⁵⁶. αβ-integrins expressed on PCa cells promote their adhesion to bone ECM proteins (e.g., collagen, fibronectin, Tenascin C), regulating cell adhesion dynamics and migratory behavior. This receptor-ligand interaction further modulates intracellular signaling cascades, thereby promoting the recruitment of additional tumor-infiltrating cells into the evolving neoplastic microenvironment. The integrin α6β4, a specialized laminin-binding integrin, exhibits frequent overexpression across diverse cancer types, with its expression level demonstrating a strong association with both malignant progression and adverse survival outcomes in oncology patients^57–59. It significantly promotes cancer cell adhesion, migration, invasion, proliferation, and tumorigenesis through activation of the Rac1, PKC, PI3K, and ERK signaling pathways, which are induced through interactions with other molecules^60,61. Our study establishes that integrin α6β4 may serve as a functional receptor for EHBP1-ssDNA, thereby implicating EHBP1-ssDNA as a pro-metastatic component of the bone microenvironment.

While FAK is a cytoplasmic non-receptor protein tyrosine kinase belonging to the protein tyrosine kinase superfamily^62,63, our data demonstrate that microenvironmental ssDNA can trigger its activation in PCa cells through integrin α6 engagement. FAK serves as a central signaling hub that integrates inputs from specific integrins, including integrin α6, and transduces them into downstream PI3K/Akt and Ras/MAPK activation^63–65, processes that are essential for tumor cell survival, migration, and metastatic colonization of bone. The functional significance of the integrin α6-FAK axis in PCa is underscored by our experimental observations: ITGA6 knockdown abolished FAK phosphorylation (Tyr397) and downstream signaling, while Defactinib blocked EHBP1-ssDNA-induced metastasis. This dual validation confirms the pathway as non-redundant and targetable.

Clinical evidence further supports the importance of this axis: FAK overexpression and hyperactivation correlate with poor prognosis across multiple solid tumors and contribute to therapeutic resistance⁶⁶. In PCa specifically, the bone metastatic niche is enriched with ssDNA fragments that can engage integrin α6 and drive FAK-dependent metastatic signaling—a mechanism that may explain the predilection of PCa for bone metastasis. Defactinib, an effective and safe oral FAK inhibitor currently being evaluated in combination therapies for pancreatic ductal adenocarcinoma⁶⁷, represents a promising therapeutic strategy for advanced PCa patients with bone metastases. Our data showing that Defactinib inhibits ssDNA-enhanced metastatic behaviors provides a strong rationale for clinical evaluation of FAK inhibitors in PCa, particularly in contexts where the integrin α6-FAK axis is hyperactivated.

In conclusion, we demonstrated the functional importance of EHBP1-ssDNA in bone marrow supernatant for promoting PCa metastasis to bone, mediated by activation of FAK signaling following underlying EHBP1-ssDNA-integrin α6β4 interactions. We also provide strong pre-clinical evidence for targeting EHBP1-ssDNA in the control of cancer and disrupting FAK signaling as a PCa metastasis therapy. Our study lays the foundation for further investigation of Defactinib as well as translational research for future clinical applications.

This study has several limitations. First, the mechanistic findings were primarily derived from mouse models and in PC-3 cell lines; validation in more animal models or even patient cohorts is needed to confirm physiological relevance. Second, while the integrin α6–FAK pathway was highlighted, the complex bone microenvironment likely involves additional factors and signaling networks that were not fully explored in this work.

Author contributions

X.C. and Y.J.-L. performed the major experiments and analyzed overall experimental data. Y.J.-L. and M.S.-Y. revised the manuscript. M.S.-Y. and Z.L.-P. assisted with the overall experiments and interpreted data. F.Y. guided the animal experiment of left ventricular injection in mice. Y.X. and Y.X. designed the experiments and revised the manuscript. Y.X. designed the experiments, curated data, and acquired funding.

Peer review

Peer review information

Communications Biology thanks Brian Thomas, Raffaella Gallo, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Johannes Stortz. A peer review file is available.

Data availability

The data that support the findings of this study are available in Supplementary Data. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD074143 (Reviewer username: reviewer_pxd074143@ebi.ac.uk, password: 71smTS4HnxaP). Unedited blot/gel images can be seen in the Supplementary Fig. 1. The gating strategy of flow cytometry can be seen in Supplementary Fig. 2.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s42003-026-09929-9.