LCAL4–FUS cooperation switches on MMP13 and drives osteolytic bone metastasis in breast cancer

Abstract

Patients with advanced breast cancer (BC) often experience bone metastasis, leading to severe skeletal complications and a significant decline in prognosis. However, the molecular mechanisms underlying BC bone metastasis remain largely unexplored. This study identified the long non-coding RNA LCAL4 as specifically upregulated in bone-metastatic BC, where it serves as an independent predictor of poor survival. Gain- and loss-of-function experiments in intracardiac and intratibial mouse models demonstrate that ectopic LCAL4 expression markedly enhances BC cell-mediated osteolytic bone metastasis. Mechanistically, LCAL4 acts as a molecular scaffold, directly binding the RNA/DNA-binding protein FUS, promoting its nuclear accumulation, and recruiting it to the MMP13 promoter. The LCAL4–FUS ribonucleoprotein complex then recruits RNA polymerase II and induces H3K4me3 deposition, thereby activating MMP13 transcription. Secreted MMP13 stimulates osteoclast differentiation and enhances bone-resorptive activity. The release of TGF-β from resorbed bone further accelerates tumor proliferation, establishing a self-reinforcing cycle that drives osteolytic metastasis. Notably, genetic disruption of the LCAL4–FUS–MMP13 pathway significantly suppresses BC bone metastasis. These findings highlight the pivotal role of the LCAL4–FUS–MMP13 axis in BC skeletal colonization and osteolytic progression, highlighting its potential as a therapeutic target for managing BC bone metastasis.

Background

Breast cancer (BC) is the most commonly diagnosed malignancy worldwide and the leading cause of cancer-related mortality among women [1]. Bone is the most frequent metastatic site in BC, with skeletal involvement observed in approximately 58% of patients with metastatic disease [2]. BC bone metastases are predominantly osteolytic (78% in one large series), with a smaller subset presenting osteoblastic or mixed phenotypes [3, 4]. Notably, 46% of patients with bone metastases subsequently experience skeletal-related events (SREs), such as hypercalcemia, pathological fractures, severe pain, and spinal cord compression, all of which significantly impair quality of life and reduce overall survival (OS) [5]. Population-based cohort data from 1999 to 2007 demonstrate a marked reduction in survival associated with bone metastasis: a five-year survival rate of 75.8% in patients without bone metastasis, 8.3% in those with bone metastasis, and only 2.5% in patients with SREs [6]. Despite this, effective therapeutic strategies for BC bone metastasis remain limited, primarily due to insufficient understanding of the underlying molecular mechanisms.

Long non-coding RNAs (lncRNAs) are RNA transcripts longer than 500 nucleotides that do not encode proteins [7]. Numerous studies have highlighted their critical roles in regulating cell differentiation, development, and various physiological processes [8]. LncRNAs interact directly with DNA, RNA, and proteins or function as scaffolds or guides for protein–DNA and protein–protein interactions, influencing chromatin architecture, splicing, transcription, protein translation, localization, and RNA processing [8, 9]. Dysregulation of lncRNAs has been linked to the initiation and progression of various human diseases, including cancer [9]. Emerging evidence implicates lncRNAs in the development of bone metastases. For example, lncRNA MAYA serves as a scaffold for the assembly of the LLGL2–MAYA–NSUN6 complex, linking the ROR1–HER3 signaling axis to the Hippo–YAP pathway, thereby promoting osteolytic bone metastases in BC [10]. Similarly, lncRNA Lnc34a epigenetically silences miR-34a expression, activating the TGF-β/Smad4 pathway and facilitating bone metastasis in hepatocellular carcinoma [11].

The lncRNA LCAL4 (LINC01614, ENSG00000230838), located at chromosomal locus 2q35, has recently been characterized and shown to be highly expressed in various human cancers, including those of the respiratory, digestive, nervous, and endocrine systems, where its upregulation is associated with poor clinical outcomes [12–15]. At the molecular level, LCAL4 acts as a decoy by binding to GSK-3β or microRNAs (e.g., miR-520a-3p and miR-217), thereby hyperactivating downstream oncogenic pathways such as WNT/β-catenin and PI3K/AKT, which enhances tumor cell proliferation, migration, and invasion [14–17]. In BC, LCAL4 expression is elevated, and its upregulation predicts shorter disease-free survival (DFS) [12, 18]. Bioinformatics analysis further reveals that high LCAL4 expression correlates with gene signatures involved in the TGF-β1 response, CDH1 signaling, and cell adhesion [18]. Silencing LCAL4 inhibits epithelial–mesenchymal transition (EMT) and increases tamoxifen sensitivity in MCF7 BC cells [19]. However, the biological role and mechanistic involvement of LCAL4 in BC bone metastasis remain largely unexplored.

In this study, LCAL4 was found to be specifically upregulated in bone-metastatic BC, correlating with poor clinical outcomes. Mechanistically, LCAL4 interacted with the RNA/DNA-binding protein FUS, promoting its nuclear translocation and anchoring it to the MMP13 promoter. The resulting LCAL4–FUS ribonucleoprotein complex recruited RNA polymerase II, facilitating H3K4me3 deposition at the MMP13 promoter and enhancing MMP13 transcription. Elevated MMP13 secretion subsequently accelerated osteoclast differentiation and activity, driving osteolytic bone metastasis. These findings reveal a regulatory mechanism governing tumor–osteoclast interactions in bone metastasis and position LCAL4 as a potential therapeutic target for BC skeletal metastases.

Materials and methods

Patients and tissue specimens

This study included 10 tumor-adjacent normal breast tissues and 132 archived paraffin-embedded BC specimens, comprising 105 primary BC tissues (63 non-metastatic, 24 bone-metastatic, and 18 non-bone-metastatic cases) and 27 BC metastases. All specimens were obtained through surgical resection or needle biopsy at Sun Yat-sen University Cancer Center, with confirmation by both clinical and pathological evaluation. Experimental procedures were approved by the Institutional Research Ethics Committee of Sun Yat-sen University. Patient specimen collection adhered to the Declaration of Helsinki, and written informed consent was obtained from all participants. Relevant clinical data are provided in Table S1 of the Supporting Information.

RNA fluorescence in situ hybridization (FISH)

LCAL4 expression in BC cells, primary, and metastatic specimens was assessed by FISH. Hybridization was used a red-labeled h-LCAL4 FISH probe mix (RiboBio, Guangzhou, China), and probe signals were visualized following the supplier’s protocols with the RiboBio FISH kit. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI), and fluorescence images were captured using an AxioVision Rel. 4.6 imaging system (Carl Zeiss, Jena, Germany).

Cell culture

Human normal mammary epithelial cell line MCF10A and BC cell lines MCF7, T47D, MDA-MB-361, BT-474, BT-549, and MDA-MB-231, along with murine BC cell line EO771 and macrophage RAW264.7 cells, were sourced from the American Type Culture Collection (ATCC, Manassas, VA, USA). The human BC cell line SUM159PT was purchased from Asterand Bioscience (Royston, UK). Human embryonic kidney HEK-293T cells were obtained from the Cell Bank of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in their recommended growth media (Procell, Wuhan, China) and underwent authentication through short tandem repeat profiling. Cells were maintained at 37 °C in a humidified incubator with 5% CO₂ and confirmed to be negative for mycoplasma contamination.

Plasmids, virus constructs, and retroviral infection

Full-length human LCAL4, MMP13, and FUS cDNAs were subcloned into the pCDH-CMV-MCS vector, and two LCAL4, MMP13, or FUS-specific shRNAs were inserted into the pLKO.1-puro vector. All constructs were provided by TransheepBio (Shanghai, China). HEK-293T cells were plated at 3 × 10⁶ cells per 10-cm dish one day before transfection. Lentiviral particles were produced by co-transfecting 6 μg of the target plasmid with 3 μg each of pCMV-dR8.2 dvpr (packaging vector, Addgene, #8455) and pCMV-VSV-G (envelope vector, Addgene, #8454), which are components of a lentiviral packaging system, using Lipofectamine 3000 (Thermo Fisher Scientific, #L3000008) according to the supplier’s protocols. Viral supernatants were harvested at 48 and 72 hours, pooled, filtered through 0.45 µm, and used to infect target cells for 72 hours. Stable cell lines were selected with 0.5 μg/ml puromycin for seven days.

To map the LCAL4-binding region of FUS, HEK-293T cells were transfected with Flag-tagged pCMV-MCS vectors encoding full-length FUS (FUS-FL) or deletion mutants FUS-∆N, FUS-∆RRM, FUS-∆Znf, and FUS-∆C using Lipofectamine 3000. At 48 hours post-transfection, cells were lysed and subjected to RNA immunoprecipitation (RIP) or RNA pull-down assays.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 9.0 (San Diego, CA, USA) and SPSS 23.0 (Chicago, IL, USA). Two-group comparisons were conducted using two-tailed unpaired Student’s t-tests, and multi-group comparisons were evaluated by analysis of variance (ANOVA), with Welch’s correction. The chi-squared test was used to assess clinical differences between high- and low-LCAL4 expression cohorts. Survival curves were generated using the Kaplan–Meier method and analyzed by the log-rank test. Univariate and multivariate analyses were conducted using Cox proportional hazards regression models to evaluate whether LCAL4 expression served as an independent prognostic factor. Linear regression was applied to assess the strength and direction of associations between variables. A P-value < 0.05 was considered statistically significant.

Results

High LCAL4 expression indicates an unfavorable bone metastasis-free survival (BMFS) outcome in patients with BC

To characterize the expression profile and clinical relevance of LCAL4 in BC, FISH was conducted on formalin-fixed, paraffin-embedded tissues. FISH analysis revealed significant enrichment of LCAL4 in bone-metastatic BC (primary tumors), with the strongest signal localized within bone lesions. In contrast, LCAL4 expression was minimal in lung-metastatic tumors and lung metastases, and nearly undetectable in normal mammary epithelium or non-metastatic primary tumors (Fig. 1A). These findings were further confirmed by qPCR, which showed elevated LCAL4 levels in bone-metastatic BC and bone metastases compared to non-metastatic primary tumors, non-bone-metastatic disease, and normal breast tissue (Fig. 1B). Clinically, high LCAL4 expression was significantly correlated with advanced disease stage and bone metastasis in patients with BC (Tables S1). Notably, elevated LCAL4 levels predicted poorer BMFS and were identified as an independent prognostic factor (Fig. 1C–E). These results were validated using data from The Cancer Genome Atlas-Breast Invasive Carcinoma (TCGA-BRCA) and curated Gene Expression Omnibus (GEO) cohorts, analyzed via the BEST platform (https://rookieutopia.com/). High LCAL4 expression was linked to reduced OS (GSE20685 and TCGA-BRCA), DFS (GSE21653), relapse-free survival (RFS, GSE20711 and TCGA-BRCA), disease-specific survival (DSS, TCGA-BRCA), and progression-free survival (PFS, TCGA-BRCA) (Fig. S1A–C). Analysis of bc-GenExMiner v5.2 data further revealed that high LCAL4 expression was associated with reduced distant metastasis-free survival (DMFS) across several subtypes, including ER all/PR all/node all (P = 0.0045), ER+/PR all/node all (P = 0.0135), ER−/PR all/node all (P = 0.0152), and ER−/PR−/node+ (P = 0.046) (Fig. S1D). These results suggest that LCAL4 upregulation plays a key role in the progression of bone metastasis in BC.

Fig. 1. High LCAL4 expression indicates an unfavorable BMFS outcome in BC patients.

A H&E staining of cellular morphology (scale bars: upper, 1 mm; lower, 100 µm) and RNA FISH analysis of LCAL4 localization and quantification (scale bar, 50 µm) in normal breast tissues (n = 10), 105 BC specimens including 63 non-metastatic, 24 bone-metastatic, and 18 other metastatic cases, as well as 27 BC metastases (15 bone metastases and 12 other metastases). B qRT-PCR analysis of LCAL4 expression in normal breast tissues and the indicated BC and metastatic samples. C Kaplan–Meier survival curves of BMFS in 105 BC patients stratified by LCAL4 expression, with P-value calculated by the log-rank test. Univariate (D) and multivariate (E) Cox regression analyses evaluating the association between LCAL4 expression and BMFS in the context of other clinical parameters (HR, hazard ratio). *P < 0.05, **P < 0.01, n.s., not significant.

LCAL4 promotes osteolytic bone metastasis and reduces survival in mice

To determine whether LCAL4 is functionally required for BC bone metastasis in vivo, stable LCAL4-overexpressing MCF7 and T47D cells (which exhibit low endogenous LCAL4 levels) were generated, and LCAL4 was silenced in the highly metastatic MDA-MB-231 line (which expresses high endogenous LCAL4) (Fig. S2A–D). A rapid bone metastasis model was established by intracardiac injection of luciferase-labeled MCF7-vector or MCF7/LCAL4 cells into estrogen-supplemented BALB/c nude mice. Longitudinal bioluminescence imaging (BLI) revealed that LCAL4 overexpression accelerated metastatic onset and significantly increased skeletal tumor burden in the hind limbs compared to vector controls (Fig. 2A, B). Micro-computed tomography (μCT) further confirmed that LCAL4 overexpression exacerbated osteolytic bone destruction (Fig. 2C). Consistent with these findings, TRAP staining and H&E of hind-limb sections demonstrated enlarged osteolytic lesions and a significant increase in TRAP-positive osteoclasts at the tumor–bone interface in the MCF7/LCAL4 group (Fig. 2D, E). By the 2-month endpoint, bone metastases developed in 83.3% of mice injected with MCF7/LCAL4 cells (verified by μCT and histology), compared to only 16.7% in the vector control group (Fig. 2F). Kaplan–Meier analysis further indicated that LCAL4 overexpression significantly reduced BMFS (Fig. 2F).

Fig. 2. LCAL4 promotes osteolytic bone metastasis and reduces survival in mice.

A Schematic of the intracardiac injection model using MCF7 cells, comparing vector-control and LCAL4-overexpressing groups (left panel), with representative BLI images of metastases (n = 6/group) (right panel). Lower panels display BLI signals restricted to hind limbs. B Quantification of normalized hind-limb BLI and fold change from baseline to endpoint (d56/d0). C Representative μCT images of bone lesions (left panel) and quantification of osteolytic area (right panel); red boxes denote osteolytic regions. Histological images (H&E and TRAP staining) (D) and quantification of tumor area and TRAP⁺ osteoclasts along the bone–tumor interface (E). Scale bar, 50 µm. F Incidence of bone metastasis (upper panel) and Kaplan–Meier BMFS curves (lower panel), with P-values calculated by log-rank test. G Schematic of the intratibial injection model using MDA-MB-231 cells, comparing Ri-vector control and LCAL4 knockdown groups (LCAL4-Ri#1 and #2). H Representative BLI and histological sections (H&E and TRAP) of hind limbs (n = 6/group). Scale bar, 50 µm. I Representative μCT images, including 3D reconstruction and axial, coronal, and sagittal views; red arrows highlight osteolytic lesions, and red ellipses mark proximal tibiae. J Quantification of hind-limb BLI signal (fold change, d35/d0), tumor area, TRAP⁺ osteoclasts, and osteolytic area. Data are denoted as mean ± SD (n = 6 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001. The schematic diagrams in A and G were created with Figdraw.

While intracardiac injection model mimics hematogenous dissemination to bone, the intratibial injection model bypasses early metastatic steps [20] and allows focused analysis of LCAL4 function in localized bone lesions and tumor-induced bone destruction. In this model, using luciferase-labeled MDA-MB-231 cells with or without stable LCAL4 knockdown, BLI revealed that LCAL4 depletion markedly reduced skeletal tumor burden in BALB/c nude mice (Fig. 2G, H, J). Corresponding μCT and H&E analyses showed significantly smaller osteolytic lesions in the hind limbs (Fig. 2H–J). TRAP staining further confirmed a significant reduction in TRAP-positive osteoclasts at the tumor–bone interface in the LCAL4-silenced group (Fig. 2H, J). To assess whether the observed phenotype is immune microenvironment-dependent, LCAL4 function was examined in an immunocompetent syngeneic model. We employed the murine BC cell line EO771 (syngeneic to C57BL/6 mice) and established luciferase-labeled cells with or without stable LCAL4 overexpression (Fig. S2E) for intratibial injection into immune-competent C57BL/6 mice. LCAL4 overexpression significantly increased skeletal tumor burden, worsened tibial osteolytic lesions, and elevated osteoclast numbers at the tumor-bone interface (Fig. S3A–D), confirming that this phenotype is not restricted to immunodeficient models.

To evaluate the therapeutic potential of targeting LCAL4, antisense oligonucleotides (ASOs), a clinically feasible nucleic acid therapeutic approach [21], were employed (Fig. S2F). In the intracardiac MDA-MB-231 xenograft model, LCAL4-ASO treatment significantly reduced the incidence of bone metastasis, skeletal tumor burden, and osteolytic lesions in the hind limbs, and TRAP-positive osteoclast numbers at the tumor-bone interface (Fig. S3E–H). These results establish LCAL4 as a critical driver of BC bone metastasis and osteolytic progression within the bone microenvironment.

Upregulation of LCAL4 in BC cells induces osteoclastogenesis

To explore the potential biological functions of LCAL4, Gene Set Enrichment Analysis (GSEA) was performed against predefined Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, Gene Ontology (GO) biological processes, and Hallmark signatures. Genes positively correlated with LCAL4 expression were significantly enriched in pathways related to extracellular matrix (ECM) remodeling, cartilage development, protein secretion, EMT, and TGF-β signaling (Fig. S4A–C), all of which are known to contribute to BC progression and metastasis [12, 19]. Given the strong pro-osteolytic phenotype induced by high LCAL4 expression in vivo, this study investigated whether LCAL4 reshapes the bone-metastatic niche by modulating osteoclast differentiation and function. Murine macrophage/pre-osteoclast RAW264.7 cells were cultured in conditioned medium (CM) derived from the corresponding BC cells. Compared to vector controls, CM from LCAL4-overexpressing cells significantly increased the number of TRAP-positive multinucleated osteoclasts (Fig. 3A, B) and TRAP enzymatic activity (Fig. 3C). In contrast, CM from LCAL4-depleted cells significantly reduced both parameters (Fig. 3A–C). Notably, ELISA quantification revealed no significant changes in PTHrP, IL-17 or TNF-α levels, which stimulate osteoblasts to secrete RANKL [22, 23], in these CM samples (Fig. S4D). Furthermore, none of the CM preparations affected the RANKL/OPG ratio in MC3T3-E1 pre-osteoblasts (Fig. S4E), indicating that LCAL4 specifically modulates the osteoclastic lineage. Additionally, several markers of osteoclast differentiation and activation, including ANPEP, PTPRE, MMP9, ACP5, and NFATC1, were significantly upregulated in RAW264.7 cells exposed to CM from LCAL4-overexpressing BC cells, whereas their expression was suppressed by CM from LCAL4-silenced cells (LCAL4-Ri#1/2) (Fig. S4F). To directly assess the functional impact of LCAL4 overexpression, RAW264.7 pre-osteoclasts were seeded on bovine cortical bone slices (Fig. 3D). The number of resorption pits per bone slice was quantified. CM from LCAL4-overexpressing BC cells significantly enhanced pit formation, while CM from LCAL4-silenced cells inhibited bone matrix resorption (Fig. 3E, F). These results suggest that LCAL4 upregulation in BC cells promotes osteoclast differentiation and enhances the bone-resorptive activity of mature osteoclasts.

Fig. 3. Upregulation of LCAL4 in BC cells induces osteoclastogenesis.

A RAW264.7 pre-osteoclasts were differentiated for 6 days with CM derived from the indicated BC cells; osteoclast formation was visualized by TRAP staining (scale bar, 10 µm). Quantification of TRAP⁺ multinucleated osteoclasts (B) and TRAP enzymatic activity (C). D Schematic of the tumor–osteoclast “vicious cycle” driven by CM from BC cells. Pre-osteoclasts were seeded on bovine bone slices and cultured with CM from the indicated BC cells; slices were then fixed, toluidine blue–stained (E), and resorption pits quantified (F) (scale bar, 100 µm). G ELISA analysis of relative TGF-β1 levels in CM from pre-osteoclasts cultured on bone slices. H CCK-8 proliferation assays of BC cells exposed to osteoclast-derived CM. Data are denoted as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Furthermore, ELISA showed that RAW264.7 pre-osteoclasts cultured on mineralized bone matrix and treated with CM from LCAL4-overexpressing BC cells released significantly more TGF-β1 than those treated with vector-control CM (Fig. 3G). This osteoclast-derived, TGF-β1-enriched CM significantly stimulated BC cell proliferation, whereas CM from LCAL4-silenced cells suppressed proliferation (Fig. 3H). These results indicate that LCAL4 upregulation in BC cells drives osteoclastogenesis and establishes a feed-forward loop that promotes tumor growth within the bone metastatic niche.

LCAL4-overexpressing BC cells secrete MMP13 to promote osteoclast differentiation and activation

To investigate the molecular mechanisms by which high LCAL4 expression promotes pre-osteoclast differentiation and maturation, three gene sets were intersected: (i) genes strongly correlated with LCAL4 expression in the TCGA-BRCA cohort, (ii) genes upregulated in bone-metastatic lesions from the GSE14017 dataset (29 distant BC metastases: 15 brain, 4 lung, 10 bone) [24], and (iii) a curated set of 20 proteases enriched at the tumor–bone interface [25]. This integrative analysis identified MMP13 and CTSK as top candidates (Fig. S5A–C). RT-PCR analysis showed that LCAL4 overexpression upregulated MMP13 in a dose-dependent manner, while CTSK expression remained unaffected in BC cells (Fig. S5D). Given MMP13’s known role in establishing bone metastatic niches [26], this gene was further examined. LCAL4 overexpression significantly increased MMP13 mRNA and protein levels in BC cells, whereas LCAL4 knockdown reduced these levels (Fig. 4A, B). Similarly, CM from LCAL4-overexpressing cells showed elevated MMP13 secretion, whereas CM from LCAL4-silenced cells had reduced MMP13 levels (Fig. 4C).

Fig. 4. LCAL4-overexpressing BC cells secrete MMP13 to promote osteoclast differentiation and activation.

A qRT-PCR analysis of relative MMP13 mRNA levels in the indicated BC cells, normalized to GAPDH. B Western blot analysis of MMP13 protein, with GAPDH as a loading control. C ELISA quantification of secreted MMP13 in CM from the indicated BC cells. V Vector, Ri-v Ri-vector, Ri#1 LCAL4-Ri#1, Ri#2 LCAL4-Ri#2. D Representative TRAP staining of multinucleated osteoclasts induced by CM from BC cells, with MMP13 knockdown (MMP13-Ri) or recombinant MMP13 (scale bar, 10 µm); quantification shown on the right. E Immunofluorescence staining of F-actin (phalloidin) revealed disrupted podosome belts after MMP13 knockdown (scale bar, 10 µm). F Bone resorption assay on bovine slices stained with toluidine blue; pit number was quantified. Scale bar, 100 µm. Data are denoted as mean ± SD of three independent experiments. **P < 0.01, ***P < 0.001.

Notably, MMP13 knockdown in BC cells abolished the capacity of CM from LCAL4-overexpressing cells to promote RAW264.7 differentiation. However, the addition of recombinant MMP13 restored the pro-differentiation effect in LCAL4-silenced CM (Fig. 4D). Podosome formation and subsequent coalescence into circumferential, actin-rich sealing zones on mineralized surfaces are essential for effective bone resorption by osteoclasts [27]. Immunofluorescence using phalloidin staining demonstrated that CM from LCAL4-overexpressing cells strongly induced peripheral F-actin–rich podosome belts in mature osteoclasts, while MMP13 knockdown suppressed actin-ring formation and inhibited pre-osteoclast fusion (Fig. 4E). In contrast, CM from LCAL4-silenced cells resulted in fragmented actin structures, which were restored by recombinant MMP13, enhancing podosome belt formation and promoting pre-osteoclast fusion (Fig. 4E). Functionally, in vitro bone resorption assays confirmed that MMP13 knockdown abrogated the bone-resorptive activity of mature osteoclasts induced by CM from LCAL4-overexpressing cells, while recombinant MMP13 rescued osteoclast activation suppressed by LCAL4-silenced CM (Fig. 4F). These results demonstrate that tumor-secreted MMP13 is essential for LCAL4-mediated regulation of osteoclast differentiation and activation.

LCAL4 interacts with FUS and facilitates its nuclear accumulation

To elucidate the mechanism by which LCAL4 upregulates MMP13 expression in BC cells, RNA pull-down coupled with mass spectrometry (MS) was performed. This analysis identified FUS, an RNA/DNA-binding protein involved in mRNA processing and transcriptional regulation [28, 29], as a binding partner of LCAL4 (Fig. 5A). Western blotting confirmed that the LCAL4 sense probe (wild-type LCAL4), but not the antisense probe, effectively retrieved endogenous FUS from MCF7 cell lysates in RNA pull-down assays (Fig. 5B). Reciprocal RIP demonstrated significant enrichment of LCAL4 in FUS immunoprecipitates compared to the IgG control (Fig. 5C). Combined immunofluorescence and RNA-FISH revealed colocalization of LCAL4 and FUS in BC cells (Fig. 5D), with markedly enhanced colocalization in tumor cells within bone metastatic lesions compared to matched primary tumors (Fig. S6A). These results suggest that the LCAL4–FUS complex is preferentially enriched in bone-metastatic tumor cells, potentially contributing to bone tropism.

Fig. 5. LCAL4 interacts with FUS and facilitates its nuclear accumulation.

A Silver-stained gel of proteins retrieved by LCAL4-sense (S) or antisense (AS) probes in RNA pull-down assays from MDA-MB-231 cells (left) and representative MS spectrum of FUS peptide (right). B Western blot validation of FUS retrieval by LCAL4-S but not LCAL4-AS probes, with GAPDH as a negative control. C RIP–qPCR analysis confirmed LCAL4 enrichment in anti-FUS immunoprecipitates relative to IgG control. D FISH and immunofluorescence revealed colocalization of LCAL4 (red) and FUS (green) in BC cells, with nuclei counterstained by DAPI (scale bar, 10 µm). E Schematic of full-length (FL) and truncated (F1-F4) LCAL4. F Western blot demonstrated that nucleotides 1245–1722 of LCAL4 are required for FUS binding. G Domain architecture of Flag-tagged FUS full-length (FL) and truncation mutants (N, N-terminus domain; RRM, RNA-recognition motif; Znf, zinc-finger motif; C, C-terminus domain). H RIP–qPCR showed that deletion of the RRM domain abolished LCAL4–FUS binding. I Flag immunoblot of LCAL4 pull-downs confirmed the necessity of the RRM domain for LCAL4 interaction, as deletion of this domain abolished binding. J Nuclear–cytoplasmic fractionation demonstrated that LCAL4 overexpression enhanced nuclear accumulation of FUS, whereas knockdown had the opposite effect. K Immunofluorescence further validated nuclear enrichment of FUS upon LCAL4 overexpression and redistribution upon knockdown (scale bar, 20 µm). L Co-IP showing increased FUS–TNPO1 interaction in LCAL4-overexpressing MCF7 cells. M Western blot of nuclear/cytoplasmic fractions showing LCAL4 promotes nuclear accumulation of wild-type FUS (WT), but not ΔNLS mutant, in MCF7 cells. H3 and GAPDH served as nuclear and cytoplasmic markers, respectively. Data are mean ± SD of three independent experiments. ***P < 0.001, n.s., not significant.

Secondary structure prediction of LCAL4 using UNAFold [30] divided the RNA into four structural fragments (Fig. S6B). Each fragment was truncated and subjected to RNA pull-down experiments (Fig. 5E). The results showed that the region spanning nucleotides 1245–1722 of LCAL4 was essential for FUS binding (Fig. 5F). To pinpoint the FUS domain mediating this interaction, a series of FUS truncation mutants were generated (Fig. 5G). Deletion of the RNA recognition motif (RRM) domain, but not other domains, completely abolished LCAL4 binding, indicating that the RRM domain of FUS is critical for this interaction (Fig. 5H, I). These data establish that nucleotides 1245–1722 of LCAL4 directly interact with the RRM domain of FUS.

Neither FUS mRNA nor total protein levels were significantly altered by LCAL4 overexpression or knockdown (Fig. S6C, D). However, nuclear and cytoplasmic fractionation followed by Western blotting revealed that LCAL4 overexpression increased nuclear FUS levels while reducing cytoplasmic expression. Conversely, LCAL4 knockdown resulted in the opposite pattern (Fig. 5J). Consistent with these findings, immunofluorescence assays demonstrated that LCAL4 overexpression concentrated FUS in the nucleus and diminished its cytosolic presence, whereas LCAL4 silencing reversed this distribution (Fig. 5K). These results suggest that LCAL4 binds FUS and facilitates its nuclear accumulation.

This study further explored how LCAL4 alters FUS subcellular localization. Previous reports indicate that FUS lacks a canonical nuclear export signal (NES) and that its nuclear export occurs through passive diffusion or alternative mechanisms independent of XPO1/CRM1-mediated active transport [31]. Thus, LCAL4 might promotes FUS nuclear import. FUS contains a proline-tyrosine nuclear localization signal (PY-NLS), which is recognized by the nuclear import receptor Transportin-1 (TNPO1) [32]. Co-IP assays demonstrated that LCAL4 overexpression enhanced the interaction between FUS and TNPO1 (Fig. 5L). Functionally, LCAL4 significantly increased the nuclear accumulation of wild-type FUS, an effect that was abolished in the FUS ΔNLS mutant, which lacks the PY-NLS domain (Fig. 5M). These results indicate that LCAL4 promotes FUS nuclear import via TNPO1 in a PY-NLS-dependent manner.

LCAL4 cooperates with FUS to transcriptionally upregulate MMP13

To assess whether FUS is essential for LCAL4-driven MMP13 upregulation, FUS was silenced in LCAL4-overexpressing MCF7 and T47D cells. The substantial increase in MMP13 mRNA and protein levels induced by LCAL4 overexpression was completely abrogated upon FUS depletion (Fig. 6A, B). Conversely, overexpressing FUS did not restore MMP13 expression that was suppressed by LCAL4 knockdown in MDA-MB-231 cells (Fig. 6A, B). Similar results were observed in luciferase reporter assays, where LCAL4 overexpression enhanced MMP13 promoter activity, which was attenuated by FUS knockdown, while enforced FUS expression failed to reverse the suppression of promoter activity induced by LCAL4 silencing (Fig. 6C). These results demonstrate that LCAL4 and FUS cooperate to drive MMP13 expression in BC.

Fig. 6. LCAL4 cooperates with FUS to transcriptionally upregulate MMP13.

A qRT-PCR showed that LCAL4-induced MMP13 expression was abolished by FUS knockdown (FUS-Ri) and was not rescued by ectopic FUS in LCAL4-depleted cells. B Western blot confirmed corresponding changes at the protein level. C Luciferase activity driven by the MMP13 promoter reporter in the indicated cells. D ChIP–qPCR revealed robust FUS occupancy at a predicted promoter-binding site (−1060 to −1054 bp), enhanced by LCAL4 overexpression and attenuated by LCAL4 knockdown. E ChIP–qPCR showed that LCAL4 increased RNA Pol II recruitment and H3K4me3 deposition at the MMP13 promoter. F Schematic model of concurrent LCAL4 and FUS binding to the MMP13 promoter. G ChIRP–Western confirmed FUS association with LCAL4 complexes. H ChIRP–PCR demonstrated LCAL4 occupancy on the MMP13 promoter region (−1317 to −1307 bp). Data are denoted as mean ± SD of three independent experiments. *P < 0.05, ***P < 0.001, n.s., not significant.

Previous studies have indicated that FUS can act as either a transcriptional activator or repressor of RNA polymerase II-mediated transcription by binding to single-stranded motifs within gene promoters [28]. Guided by this model, a potential FUS-binding site was identified at −1060 to −1054 in the MMP13 promoter (Fig. 6D). ChIP–qPCR confirmed robust FUS occupancy at this locus in MCF7, T47D, and MDA-MB-231 cells, with this enrichment further enhanced by LCAL4 overexpression and diminished following LCAL4 knockdown (Fig. 6D). In parallel, LCAL4 upregulation increased RNA polymerase II recruitment and H3K4me3 deposition at the MMP13 promoter, whereas LCAL4 silencing reduced both marks (Fig. 6E). These results indicate that LCAL4 facilitates FUS binding at the MMP13 promoter, thereby promoting transcriptional activation.

To investigate whether LCAL4 directly associates with the MMP13 promoter, in-silico sequence complementarity analyses were performed using Gaemons [33] and the UCSC Genome Browser [34]. An 11-nucleotide segment (−1317 to −1307) within the MMP13 promoter was identified as the most probable LCAL4-binding site (Fig. 6F). Chromatin isolation by RNA purification (ChIRP), followed by immunoblotting and qPCR, confirmed the physical interaction between LCAL4 and FUS and corroborated LCAL4 occupancy at the predicted MMP13 promoter region (−1317 to −1307 bp) (Fig. 6G, H). These results establish LCAL4 as a molecular scaffold that anchors the MMP13 promoter and recruits FUS, thereby facilitating chromatin remodeling and transcriptional activation of MMP13.

Targeting FUS or MMP13 blocks LCAL4-mediated osteolytic metastasis

Next, the role of the FUS–MMP13 axis in LCAL4-driven osteolytic bone metastasis was evaluated. In vitro osteoclast differentiation assays showed that silencing FUS abolished the stimulatory effect of CM from LCAL4-overexpressing cells on RAW264.7 differentiation. Conversely, ectopic FUS expression in LCAL4-depleted BC cells did not significantly increase the number of TRAP-positive multinucleated osteoclasts (Fig. 7A). Similarly, bone resorption assays revealed that FUS knockdown eliminated the enhanced bone-resorbing activity of mature osteoclasts stimulated by CM from LCAL4-overexpressing cells, while enforced FUS expression did not restore osteoclast activity suppressed by LCAL4 depletion (Fig. 7B).

Fig. 7. Targeting FUS or MMP13 blocks LCAL4-mediated osteolytic metastasis.

A TRAP staining showed that silencing FUS abolished the pro-osteoclastogenic effects of CM from LCAL4-overexpressing BC cells, while enforced FUS expression in LCAL4-depleted cells failed to rescue osteoclast differentiation (scale bar, 10 µm). B Bone resorption assays demonstrated that FUS knockdown eliminated LCAL4-induced osteoclast resorptive activity, and enforced FUS failed to restore it (scale bar, 100 µm). C In vivo experiments using MCF7/LCAL4 cells with or without FUS/MMP13 knockdown showed reduced hind-limb tumor burden and osteolytic lesions in the depletion groups, as demonstrated by BLI and histology (H&E, TRAP; scale bar, 50 µm). D Quantification of hind-limb BLI signal (fold change, d56/d0), tumor area, and TRAP⁺ osteoclasts. E μCT imaging demonstrated reduced osteolytic areas following FUS or MMP13 silencing. F Incidence of bone metastasis (upper panel) and Kaplan–Meier BMFS curves (lower panel). P-value calculated by log-rank test. Data are denoted as mean ± SD (n = 6 mice per group). *P < 0.05, **P < 0.01, n.s., not significant.

The in vivo impact of FUS or MMP13 inhibition on BC bone metastasis was then assessed. Highly bone-metastatic MCF7/LCAL4 cells, with or without concurrent FUS or MMP13 knockdown, were injected into the cardiac ventricle of nude mice. Silencing either gene significantly reduced hind-limb tumor burden and osteolytic lesions compared to scramble controls (Fig. 7C–E). TRAP staining further confirmed a marked reduction in osteoclast numbers at the tumor–bone interface in both knockdown groups (Fig. 7C, D). Moreover, depletion of FUS or MMP13 in LCAL4-overexpressing BC cells decreased the incidence of bone metastasis, delayed metastatic onset, and significantly prolonged BMFS (Fig. 7F). These results demonstrate that both MMP13 and FUS are indispensable mediators of LCAL4-driven osteolytic bone metastasis in BC.

Clinical relevance of the LCAL4–FUS–MMP13 axis in human BC

To evaluate the clinical relevance of the LCAL4–FUS–MMP13 axis identified in vitro and in vivo, FISH and IHC were conducted on archived, formalin-fixed tissue samples of non-metastatic BC (n = 63), bone-metastatic BC (n = 24), and bone metastases (n = 15). MMP13 expression showed a progressive increase across non-metastatic BC, bone-metastatic BC, and bone metastasis tissues, paralleling the trend observed for LCAL4 expression in Fig. 1A, B. In contrast, FUS exhibited a modest, non-significant increase (Fig. 8A). These results were corroborated by qRT-PCR analysis of non-bone-metastatic and bone-metastatic BC tissue samples (Fig. 8B). Correlation analysis further demonstrated LCAL4 expression was significantly correlated with MMP13 (r = 0.455, P < 0.001), and FUS expression also showed a significant correlation with MMP13 (r = 0.338, P < 0.001). However, no statistically significant correlation was found between LCAL4 and FUS (r = 0.113, P = 0.252) (Fig. 8C–E). These findings support the LCAL4–FUS–MMP13 axis as a key regulatory network promoting tumor–bone stromal interactions during metastatic progression (Fig. 8F).

Fig. 8. Clinical relevance of the LCAL4–FUS–MMP13 axis in human BC.

A Left: Representative FISH images for LCAL4 (scale bar, 50 µm), IHC staining for FUS and MMP13 (scale bar, 100 µm), and H&E staining (scale bar, 100 µm) in the indicated tissues. Right: Violin plots showing expression levels of FUS and MMP13 (IHC H-scores) in non-metastatic BC (n = 63), bone-metastatic BC (n = 24), and bone metastasis samples (n = 15). *P < 0.05, **P < 0.01, n.s., not significant. B Expression levels of LCAL4, FUS, and MMP13 determined by qRT-PCR in non-bone-metastatic BC (n = 81) and bone-metastatic BC (n = 24), shown as box plots. Linear regression analyses revealed significant correlations between LCAL4 and MMP13 (C), as well as FUS and MMP13 (D), but not between LCAL4 and FUS (E). F Schematic model illustrating (created with Figdraw) that LCAL4 binds FUS, promotes its nuclear localization, and transcriptionally upregulates MMP13, which induces pre-osteoclast differentiation and maturation, ultimately driving osteolytic bone metastasis in BC.

Discussion

LncRNA LCAL4 has been widely recognized as an oncogenic driver and a poor prognostic marker in various human malignancies [13, 16, 18]. Its expression is significantly elevated in most tumor types, except for certain cancers of the reproductive and urinary systems [13]. Functionally, LCAL4 promotes malignant progression by enhancing cancer cell proliferation, invasion, EMT, and chemoresistance [15, 19]. Nevertheless, its role in BC bone metastasis remained unclear. This study demonstrates that LCAL4 is markedly upregulated in bone-metastatic BC and correlates with shorter BMFS. In line with previous reports, LCAL4 expression was higher in hormone receptor-positive (HR + ) and HER2+ tumors compared to triple-negative BC (TNBC) [18, 35]. Elevated LCAL4 levels were also associated with reduced DMFS across various molecular subtypes. Notably, both gain- and loss-of-function experiments in luminal A (MCF7/T47D) and TNBC (MDA-MB-231) models confirmed that LCAL4 promotes osteoclastogenesis and osteolytic bone metastasis, irrespective of subtype, highlighting the conserved pro-metastatic role of LCAL4 despite variable baseline expression. Mechanistically, LCAL4 facilitates osteoclastogenesis via paracrine secretion of MMP13 from tumor cells, which accelerates bone matrix degradation. The subsequent release of TGF-β1 from the bone matrix further drives tumor expansion and osteolytic metastasis. Notably, targeting LCAL4 with specific shRNAs or ASOs significantly inhibited bone metastasis and osteolytic lesion formation in vivo. These findings unveil a previously unrecognized role of LCAL4 in BC bone metastasis and suggest its potential as both a prognostic biomarker and a therapeutic target in bone-metastatic BC.

FUS is a DNA/RNA-binding protein originally identified as a fusion oncogene in human liposarcomas [36]. Point mutations in the FUS gene have also been linked to neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) [37]. FUS dynamically shuttles between the cytoplasm and nucleus. In addition to orchestrating DNA damage repair, mRNA maturation, and alternative splicing [29, 38, 39], FUS functions as a chromatin-associated transcriptional regulator [39]. It cooperates with nuclear hormone receptors [40] and gene-specific transcription factors [41] to regulate target gene transcription. Additionally, it modulates RNA polymerase II- and III-driven transcription through direct interactions with their respective subunits [42, 43]. Moreover, FUS suppresses R-loop accumulation during transcription and restrains hyperphosphorylation of Ser2 within the RNA polymerase II C-terminal domain, thereby maintaining transcriptional homeostasis [39, 44]. Depending on the cellular context, FUS can either activate or repress RNA polymerase II-transcribed genes by binding to specific single-stranded promoter motifs [28]. In the present study, FUS was identified as a direct transcriptional activator of MMP13 and as a downstream effector of lncRNA LCAL4, binding to the MMP13 locus to enhance its expression. Given the inherently pleiotropic nature of FUS-mediated transcriptional regulation, further investigation is needed to determine whether FUS cooperates with additional transcription factors or cofactors in regulating MMP13 expression.

Osteolytic bone metastasis is driven by osteoclast hyperactivation and the release of osteolytic factors from tumor and stromal cells at resorption sites [26, 45]. MMP13 (collagenase-3) degrades collagens I–III and other ECM components, including the bone matrix [46]. This proteolytic activity remodels the ECM, releasing sequestered growth factors that facilitate pre-metastatic niche formation [47–49]. Elevated MMP13 expression is observed in various cancers [50], including BC, where it promotes tumor proliferation, angiogenesis, migration, and invasion [48, 51, 52]. In addition to these tumor-intrinsic effects, MMP13 enhances osteoclast differentiation by activating pro-MMP9 and cleaving galectin-3, an inhibitor of osteoclastogenesis, thereby promoting BC bone metastasis [26]. However, emerging evidence suggests that MMP13 can also stimulate osteoclastogenesis independently of its catalytic activity by engaging PD-1H on osteoclast progenitors and activating the ERK1/2–NFATc1–DC-STAMP signaling pathway [53, 54]. This dual mechanism implies that catalytic inhibitors alone may not sufficiently block MMP13-mediated osteoclastogenesis and bone resorption.

This study delineates a transcriptional circuit in which LCAL4 functions as a scaffold, promoting FUS nuclear accumulation and recruitment to the MMP13 promoter, thereby inducing chromatin remodeling and transcriptional activation. The secreted MMP13 subsequently drives osteoclastogenesis and bone resorption in distant skeletal lesions. Notably, LCAL4 expression does not influence levels of canonical osteolytic factors (PTHrP, IL-17, TNF-α) or the RANKL/OPG ratio in pre-osteoblasts, indicating that this pathway operates independently of RANKL/RANK signaling. These findings reveal a previously unrecognized mechanism regulating MMP13 expression in BC and highlight the LCAL4–MMP13 axis as a complementary therapeutic target. Combining targeting of this axis with anti-RANKL agents (e.g., denosumab) or bisphosphonates could provide additive inhibition of osteoclastogenesis.

Our study establishes the LCAL4–FUS–MMP13 axis as a tumor-intrinsic driver of osteolytic bone metastasis in BC. Pharmacological inhibition of this axis presents a promising therapeutic strategy for preventing bone metastasis. Potential druggable targets include ASOs directed at LCAL4, FUS, or MMP13. ASO-mediated LCAL4 silencing demonstrated proof-of-concept efficacy in our preclinical model, and ASOs are well-established nucleic acid therapeutics with proven clinical success [21]. The development of FUS-targeting therapeutics, such as Jacifusen (Ulefnersen/ION363), a first-in-class ASO currently in Phase 3 trials for ALS (NCT04768972) [55], further supports FUS as a druggable target and strengthens the translational potential of this strategy for bone-metastatic BC. Additional approaches include small-molecule disruptors of the LCAL4–FUS interaction, which block FUS nuclear accumulation and recruitment to the MMP13 promoter, as well as agents targeting MMP13 expression or function. However, the clinical efficacy of these strategies remains to be validated in future studies.

Despite these advances, several questions remain. First, how the LCAL4 axis is regulated by pro-osteoclastogenic factors (e.g., RANKL, M-CSF) under physiological conditions is still unclear. Second, further investigation is needed to determine whether the LCAL4–FUS complex recruits chromatin-modifying enzymes or additional transcriptional cofactors for MMP13 transcriptional regulation. Third, unlike the osteoclast-intrinsic tumor-suppressive role of lncRNA Malat1, which inhibits NFATC1 activation in pre-osteoclasts to restrain bone metastasis [56], it is unclear whether LCAL4 is dysregulated in bone microenvironment cells such as osteoclast precursors or immune cells under pathological conditions. Additionally, it remains to be determined whether stromal- or immune-derived LCAL4 contributes to physiological bone remodeling or metastatic niche formation.

Author contributions

QL, MF, XS, and XY were responsible for all data collection and analysis. YZ and ZZ performed molecular, cellular, and animal experiments. Xuan Z, Xian Z, and QZ performed biochemical experiments. ZX is responsible for collecting clinical samples and conducting statistics. KL, CZ, and QT provided administrative support. LY, MW, and YW supervised the project. QL and LY wrote the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by grants from the National Natural Science Foundation of China (82473046, 82372907, 82303501, 82202850, 82404676), Guangdong Basic and Applied Basic Research Foundation (2025A1515012462, 2023A1515030058, 2023A1515110993, 2025A1515012797), Shenzhen Science and Technology Program (JCYJ20210324121809026, JCYJ20250604143508011, JCYJ20250604143522028, JCYJ20230807110701003, RCYX20210706092141082, JCYJ20230807110306013, JCYJ20220530144817040), Research start-up fund of part-time PI, SAHSYSU (ZSQYJZPI202008).

Data availability

Data are available upon request from the authors.

Competing interests

The authors declare no competing interests.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

The use of clinical specimens in this study was approved by the Institutional Research Ethics Committee of Sun Yat-sen University, with informed consent obtained from all patients prior to sample collection. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University and were conducted in strict accordance with relevant guidelines and regulations.

Supplementary information

The online version contains supplementary material available at 10.1038/s41388-026-03848-z.