Intracellular sclerostin promotes tumor progression and metastasis as a potential therapeutic target in triple-negative breast cancer

Summary

Triple-negative breast cancer (TNBC) urgently requires promising therapeutic targets. This study identifies sclerostin, an osteocyte-derived secretory protein traditionally linked to bone homeostasis, as an unexpected intracellular oncogenic driver in TNBC. Although genetic ablation of sclerostin markedly suppresses tumor progression and lung metastasis, neither its antibody nor recombinant protein exerts any effects, excluding the role of extracellular sclerostin in TNBC. Genetic and pharmacological approaches (sclerostin aptamer-based proteolysis-targeting chimera with potent intracellular sclerostin-degrading activity, Apc101) show the emerging role of intracellular sclerostin in promoting TNBC progression and metastasis. Notably, in both TNBC cell-derived and patient-derived xenograft models, Apc101 significantly suppresses tumor progression. Mechanistically, intracellular sclerostin interacts with caprin1 to stabilize CDK1 and Cyclin B1 mRNAs. Collectively, this study reveals an oncogenic function of intracellular sclerostin in TNBC and proposes that targeting it represents a promising therapeutic strategy.

Graphical abstract

Highlights

• •This study reveals a crucial role of intracellular sclerostin in TNBC
• •This study advances the target understanding of sclerostin beyond its extracellular roles
• •Sclerostin-caprin1 interaction is crucial for promoting TNBC progression and metastasis
• •Targeting intracellular sclerostin might represent a promising therapeutic strategy

Sun et al. reveal that intracellular sclerostin plays a crucial role in promoting TNBC progression and metastasis. Mechanistically, intracellular sclerostin interacts with caprin1 to stabilize CDK1 and Cyclin B1 mRNAs. Targeting intracellular sclerostin might represent a promising therapeutic strategy, particularly for patients who currently lack effective treatment options.

Introduction

The International Agency for Research on Cancer has released a pivotal report highlighting an alarming trajectory: if the current trends persist, global breast cancer cases could surge by 38%, while deaths linked to the disease may increase by 68% by 2050.¹ As the most aggressive subtype, triple-negative breast cancer (TNBC) exhibits significant heterogeneity, frequent metastasis and recurrence, and poor outcomes. Currently, chemotherapy with severe toxicity remains the primary treatment option.² Despite the extensive development of molecular targets, clinical response rates remain unsatisfactory. Pembrolizumab (a PD-1 inhibitor) is an FDA-approved targeted therapy for advanced TNBC with PD-L1-expressing tumors (combined positive score ≥10). However, only 38.1% of patients (323 of 847) met this criterion, indicating that just a small subset could potentially benefit from pembrolizumab combined with chemotherapy compared with chemotherapy alone.³ In contrast, sacituzumab govitecan, a Trop-2-directed antibody-drug conjugate, showed efficacy across all Trop-2 expression levels in the phase 3 ASCENT trial.⁴ However, Trop-2 expression levels strongly influenced outcomes: patients with weak/absent expression had significantly shorter progression-free survival than those with moderate-to-strong expression (3.1 vs. 7.1 months; p = 0.019).⁵ Therefore, there is a critical unmet need to identify promising molecular targets that could benefit most TNBC patients.

Sclerostin, an osteocyte-derived secretory protein encoded by SOST gene, acts as a critical brake on bone formation.⁶ The FDA has approved a humanized anti-sclerostin antibody (Scl-Ab) as a therapeutic intervention for postmenopausal osteoporosis. Traditionally, sclerostin has been characterized as an inhibitor of Wnt signaling, a pathway whose activation has been demonstrated to accelerate tumor development.⁷ Thus, sclerostin was initially proposed to exhibit anti-tumor activity by inhibiting the Wnt signaling pathway.

Intriguingly, while SOST mRNA is absent in healthy breast tissue, it is expressed in 56% of clinical TNBC specimens.⁸ And another study reported a negative correlation between SOST mRNA expression and disease-free survival based on the public The Cancer Genome Atlas database, indicating the potential of sclerostin as a prognostic indicator for TNBC.⁹ Surprisingly, although sclerostin’s Wnt-inhibitory function would theoretically suppress tumors, Scl-Ab was reported to show no anti-tumor effects in TNBC mouse models.¹⁰ In contrast, a small-molecule sclerostin inhibitor was reported to inhibit TNBC proliferation in vitro.⁹ This discrepancy might stem from a key difference between antibodies and small-molecule inhibitors: unlike sclerostin antibodies, small-molecule inhibitors could be internalized into cells to target intracellular sclerostin, potentially indicating its pro-tumor function. These contrasting outcomes sparked our interest in investigating the role of intracellular sclerostin in TNBC.

Here, our clinical TNBC biobank analysis further confirmed sclerostin protein expression in a substantial proportion of TNBC specimens, correlating with elevated level of tumor proliferation marker. In our TNBC xenograft and syngeneic models, sclerostin knockout in TNBC cells markedly impeded tumor progression and lung metastasis, highlighting its critical role in TNBC. Interestingly, neither recombinant sclerostin protein nor Scl-Ab had any effect on TNBC progression both in vitro and in vivo, excluding the role of extracellular and systemic sclerostin. Genetic and pharmacological approaches (sclerostin aptamer-based proteolysis-targeting chimera [PROTAC], Apc101) show the emerging role of intracellular sclerostin in promoting TNBC progression and metastasis. Remarkably, in a sclerostin-positive TNBC patient-derived xenograft (PDX) model, Apc101 significantly suppressed tumor progression. This indicates that targeting intracellular sclerostin might represent a promising therapeutic strategy for patients who currently lack effective treatment options. In the pull-down assay, we identified caprin1 as an interactive sclerostin protein in TNBC cells. Caprin1, an RNA-binding oncoprotein to regulate cell cycle, was reported to stabilize STAT1 mRNA¹¹ and to promote tumor progression and metastasis in various cancers, including breast cancer.^12,13,14 In TNBC, we found that the interaction between intracellular sclerostin and caprin1 is required by intracellular sclerostin to stabilize CDK1 and Cyclin B1 mRNAs and drive tumor progression and metastasis.

Results

The clinical features of sclerostin within tumor tissues from TNBC patients

To evaluate sclerostin protein expression in clinical breast cancer tissues, we quantified sclerostin levels using immunohistochemistry (IHC)-based H-scoring in our clinical cohort comprising 100 TNBC, 94 HER2-positive, and 99 luminal cases. Interestingly, sclerostin protein demonstrated differential expression patterns across breast cancer subtypes (Figure 1A). Detectable sclerostin levels (defined as H-score > 50) were observed in 60% of TNBC specimens, in contrast to only 9% of HER2-positive and 8% of luminal cases (Figure 1B). To investigate the clinical significance of this subtype-specific expression pattern, we analyzed the correlation between sclerostin and clinical tumor proliferation marker Ki-67 in TNBC tissues. A robust positive correlation was observed (Figure 1C). Consistently, pooled correlation analysis of publicly available mRNA expression datasets revealed a statistically significant positive correlation between SOST and MKI67 (Ki-67) mRNA expression (Pearson r = 0.571, p < 0.0001) (Figure S1). These findings suggest that sclerostin might serve as an important biomarker for TNBC malignancy.

Figure 1.

The clinical features of sclerostin within tumor tissues from TNBC patients

(A) Representative immunohistochemistry (IHC) images of sclerostin-positive (H-score > 50)/-negative (H-score < 50) tumor tissues from TNBC/HER2/luminal patients in our clinical breast cancer biobank (upper). Individual H-score distributions for TNBC/HER2/luminal subtypes with density histograms and fitted normal distribution curves (black lines) (lower). Red dashed lines mark the cutoff at H-score = 50, with red-shaded areas indicating positive cases. Statistical parameters (μ, mean; σ, standard deviation) and positivity rates are displayed for each subtype (n = 100 for TNBC; n = 94 for HER2; n = 99 for luminal). Scale bars, 100 μm.

(B) Proportion for sclerostin-positive (H-score > 50)/-negative (H-score < 50) tumor tissues from TNBC/HER2/luminal patients in our clinical breast cancer biobank.

(C) Correlation analysis for the protein expression of sclerostin and Ki-67 within clinical TNBC tissues (left), and representative IHC images for sclerostin-positive case 1 and sclerostin-negative case 2 (right). p value was determined by Spearman’s rank correlation analysis. Scale bars, 100 μm.

The role of sclerostin in TNBC progression and metastasis both in vitro and in vivo

To explore the role of sclerostin in human TNBC cells in vitro, colony formation, cell growth, and transwell assays were performed on mCherry-expressing wild-type (WT) MDA-MB-231 (231-SOST ^WT) cells and mCherry-expressing sclerostin-knockout MDA-MB-231 (231-SOST ^KO) cells. In the colony formation and cell growth assays, statistical data showed that both the colony formation and the cell growth rate were significantly lower in 231-SOST ^KO than in 231-SOST ^WT cells (Figure 2A). In the transwell assay, the statistical data showed that the migration and invasion rates were significantly lower in 231-SOST ^KO than in 231-SOST ^WT cells (Figure 2B). Consistent findings were observed in murine TNBC 4T1 cells (Figures 2F and 2G). It suggests a crucial role of sclerostin in TNBC proliferation, migration, and invasion in vitro.

Figure 2.

The role of sclerostin in proliferation, migration, and invasion in TNBC cells in vitro and progression and metastasis in TNBC cell-inoculated orthotopic and metastatic mouse models

(A) Representative images of colony formation (left), and statistics for comparing relative colonies % (middle) and cell growth rate (right) between mCherry-expressing wild-type MDA-MB-231 (231-SOST^WT) cells and mCherry-expressing SOST-knockout-1 MDA-MB-231 (231-SOST^KO-1) cells/mCherry-expressing SOST-knockout-2 MDA-MB-231 (231-SOST^KO-2) cells.

(B) Representative images of migration and invasion (left), and statistics for comparing relative migration % (upper) and invasion % (lower) between 231-SOST^WT and 231-SOST^KO-1/231-SOST^KO-2 cells. Scale bars, 200 μm.

(C) Representative fluorescence images of tumor-bearing mice (left), and statistics for comparing tumor fluorescence intensity at primary site between 231-SOST^WT and 231-SOST^KO-1 (231-SOST^KO) cells-inoculated orthotopic mouse models (n = 6 for each group) (right).

(D) Representative images of primary tumors (left), and statistics for comparing tumor growth rate between 231-SOST^WT and 231-SOST^KO cells-inoculated orthotopic mouse models (n = 6 for each group) (right).

(E) Representative hematoxylin-and-eosin images of lung tissues (left), and statistics for comparing lung metastatic nodule number between 231-SOST^WT and 231-SOST^KO cells-inoculated metastatic mouse models (n = 5 for each group) (right). Scale bars, 1 mm.

(F) Representative images of colony formation (left) and statistics for comparing relative colonies % (middle) and cell growth rate (right) between luciferase reporter-expressing wild-type 4T1 (4T1-sost^WT) cells and luciferase reporter-expressing sost-knockout-1 4T1 (4T1-sost^KO-1) cells/luciferase reporter-expressing sost-knockout-2 4T1 (4T1-sost^KO-2) cells.

(G) Representative images of migration and invasion (left), and statistics for comparing relative migration % (upper) and invasion % (lower) between 4T1-sost^WT and 4T1-sost^KO-1/4T1-sost^KO-2 cells. Scale bars, 200 μm.

(H) Representative bioluminescent images of tumor-bearing mice (left), and statistics for comparing tumor bioluminescent intensity at primary site between 4T1-sost^WT and 4T1-sost^KO-1 (4T1-sost^KO) cells-inoculated orthotopic mouse models (n = 6 for each group) (right).

(I) Representative images of primary tumors (left), and primary tumor growth curve for comparing tumor growth rate between 4T1-sost^WT and 4T1-sost^KO cells-inoculated orthotopic mouse models (n = 6 for each group) (right).

(J) Representative bioluminescent images of lung tissues (left), and statistics for comparing tumor bioluminescent intensity in lung tissues between 4T1-sost^WT and 4T1-sost^KO cells-inoculated metastatic mouse models (n = 5 for each group) (right).

Data are presented as mean ± SD. For in vitro studies, error bars represent three independent experiments. For in vivo studies, each spot represents one subject. For comparing colony formation, migration, and invasion (A, B, F, and G), p value was determined by one-way ANOVA with Dunnett post hoc test. For comparing total flux and number of lung metastatic nodules (C, E, H, and J), p value was determined by unpaired t test. For comparing cell growth rate (A and F), p value was determined by two-way ANOVA with Dunnett post hoc test. For comparing tumor growth rate (D and I), p value was determined by two-way ANOVA with Bonferroni post hoc test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

To explore the role of sclerostin in human TNBC progression in vivo, 231-SOST ^WT and 231-SOST ^KO cells-inoculated orthotopic mouse models were established. Fluorescence images of the tumor-bearing mice were captured to trace tumor progression (Figure 2C, left). Statistical data showed that the fluorescence intensity of primary tumors was significantly lower in 231-SOST ^KO orthotopic mice than in 231-SOST ^WT orthotopic mice (Figure 2C, right). Both the tumor images and the statistical data for tumor volume measurement showed that the volume of primary tumors was significantly smaller in 231-SOST ^KO orthotopic mice than in 231-SOST ^WT orthotopic mice (Figure 2D). Consistent findings have also been observed in 4T1 mouse models (Figures 2H and 2I). This suggests a crucial role of sclerostin in TNBC progression in vivo.

To explore the role of sclerostin in human TNBC metastasis in vivo, 231-SOST ^WT and 231-SOST ^KO cells-inoculated metastatic mouse models were established. Compared with 231-SOST ^KO metastatic mice, obvious metastatic nodules in lung tissues were observed in 231-SOST ^WT metastatic mice (Figure 2E, left). Statistical data showed that the number of lung metastatic nodules was significantly lower in 231-SOST ^KO metastatic mice than in 231-SOST ^WT metastatic mice (Figure 2E, right). Consistent findings have also been observed in 4T1 mouse models (Figure 2J). This suggests a crucial role of sclerostin in TNBC metastasis in vivo.

Together, it suggests a crucial role of sclerostin in TNBC progression and metastasis both in vitro and in vivo.

The role of extracellular and systemic sclerostin could be excluded in TNBC

To investigate the effect of sclerostin antibody (Scl-Ab) with high cross-reactivity in both human and mouse sclerostin¹⁵ on TNBC cells in vitro, colony formation, cell growth, and transwell assays were conducted on 231-SOST ^WT and 4T1-sost ^WT cells, respectively. No differences were found in colony formation and cell growth rate, as well as migration and invasion rates between the IgG control and Scl-Ab groups (Figure S2). Moreover, confocal microscopy data showed that there was nearly undetectable fluorescein signal in TNBC cells treated with fluorescein-labeled Scl-Ab (Figure S3), indicating the negligible cellular internalization ability of Scl-Ab in TNBC cells.

To explore the effect of human recombinant sclerostin protein (h-rsost) on human TNBC cells in vitro, colony formation, cell growth, and transwell assays were conducted on both 231-SOST ^WT and 231-SOST ^KO cells. No differences were found in colony formation and cell growth rate, as well as migration and invasion rates, between the PBS and h-rsost groups in both 231-SOST ^WT (Figures S4A and S4C) and 231-SOST ^KO cells (Figures S5A and S5C). Consistent findings were observed in 4T1-sost ^WT (Figure S4B and S4D) and 4T1-sost ^KO cells (Figures S5B and S5D). It indicates that the role of extracellular sclerostin in TNBC could be excluded in vitro.

Pharmacologically, to explore the effect of Scl-Ab on TNBC progression in vivo, 4T1 cell-inoculated orthotopic mouse model was established. No differences were found in tumor weight and volume between the IgG control and Scl-Ab groups (Figure S6A). Genetically, to explore the effect of systemic sclerostin depletion on TNBC progression in vivo, 4T1 cell-inoculated orthotopic WT mice and sclerostin-knockout (SOST ^−/−) mice were established. No differences were found in tumor weight and volume between 4T1 cell-inoculated orthotopic WT mice and SOST ^−/− mice (Figure S6B). The above pharmacological and genetic evidence indicates that the role of systemic sclerostin in TNBC could be excluded in vivo.

The role of intracellular sclerostin in TNBC proliferation and migration in vitro

Genetically, we constructed transgenic TNBC cell lines that only produced non-secreted intracellular sclerostin (231-SOST ^Intra and 4T1-sost ^Intra) as described previously¹⁶ (Figure S7). The statistical data showed that the colony formation and migration rates were significantly higher in 231-SOST ^Intra than in 231-SOST ^KO and 231-SOST ^WT cells (Figures 3A and 3B). Consistent findings were also observed in 4T1 cells (Figures S11A and S11B). The above genetic evidence suggests a crucial oncogenic role of intracellular sclerostin in TNBC in vitro.

Figure 3.

The role of intracellular sclerostin in TNBC proliferation and migration in MDA-MB-231 cells in vitro

(A and B) Representative images of colony formation and statistics for comparing relative colonies % (A) as well as representative images of migration and statistics for comparing relative migration % (B) among 231-SOST^WT, 231-SOST^Intra, and 231-SOST^KO cells. Scale bars, 200 μm.

(C) Western blot images of intracellular sclerostin level in MDA-MB-231 cells treated with PBS, Apc001OA (0.35 μM), and sclerostin aptamer-based PROTACs with different E3 ligands (0.35 μM) for 6 h.

(D) Western blot images of intracellular sclerostin level in MDA-MB-231 cells treated with PBS and sclerostin aptamer-based PROTACs with different linkers (0.35 μM) for 6 h.

(E) SPR analysis of sclerostin/Apc101/VHL ternary complex.

(F) Confocal fluorescence microscopy images of MDA-MB-231 cells treated with PBS, Cy3-labeled scramble (0.35 μM), and Cy3-labeled Apc101 (0.35 μM) for 3 h.

(G) Western blot images of intracellular sclerostin level in MDA-MB-231 cells treated with PBS and Apc101 with different concentrations (0.05, 0.1, 0.3, 0.5, 0.7 μM) for 6 h.

(H) The dose-response curve of intracellular sclerostin degradation effect of Apc101.

(I) Western blot images of intracellular sclerostin level in MDA-MB-231 cells treated with Apc101 for different durations (0, 3, 6, 12, and 24 h).

(J) Western blot images of intracellular sclerostin level in MDA-MB-231 cells treated with PBS, Apc101 (0.7 μM), MLN4924 (ubiquitination inhibitor) (1 μM), MG132 (proteasome inhibitor) (1 μM), Apc101ˆ (negative control compound) (0.7 μM), Apc101 (0.7 μM) in the presence of MLN4924 (1 μM), and Apc101 (0.7 μM) in the presence of MG132 (1 μM).

(K and L) Representative images of colony formation and statistics for comparing relative colonies % (K) as well as representative images of migration and statistics for comparing relative migration % (L) among PBS, Apc101 (0.7 μM), and Apc101ˆ (0.7 μM) groups in MDA-MB-231 cells. Scale bars, 200 μm.

Data are presented as mean ± SD. For in vitro studies, error bars represent three independent experiments. For comparing colony formation and migration (A and B), p value was determined by one-way ANOVA with Tukey post hoc test. For comparing colony formation and migration (K and L), p value was determined by one-way ANOVA with Dunnett post hoc test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Pharmacologically, we designed sclerostin aptamer-based PROTACs to degrade intracellular sclerostin. The sclerostin aptamer-based PROTAC consisted of three components: the sclerostin aptamer, an E3 ligand, and a linker. For aptamer, Apc001 was screened against sclerostin by Systematic Evolution of Ligands by Exponential Enrichment (SELEX), which exhibited the highest binding affinity (KD = 43.1 nM) among all candidates.⁶ Moreover, octadecanedioic acid (OA) was conjugated with Apc001 to prolong the half-life of conjugated Apc001 (Apc001OA).^17,18 To investigate the effect of E3 ligand on the degradation activity of PROTAC, we synthesized sclerostin aptamer-based PROTACs with different E3 ligands (Apc001OA-P4-CRBN, Apc001OA-P4-VHL, and Apc001OA-P4-MDM2) by conjugating 5′-alkyne-modified Apc001OA with azide-linked E3 ligands (Figure S8). Our western blot data showed that the intracellular sclerostin level was lower in the Apc001OA-P4-VHL group compared with the other groups in MDA-MB-231 cells in vitro (Figure 3C). To investigate the effect of linker on the degradation activity of PROTAC, we synthesized a series of sclerostin aptamer-based PROTACs with different PEG linkers (Apc001OA-P2-VHL, Apc001OA-P4-VHL, Apc001OA-P6-VHL, Apc001OA-P8-VHL, and Apc001OA-P10-VHL) (Figure S9). Our western blot data demonstrated that the intracellular sclerostin level was lowest in the Apc001OA-P4-VHL group among all candidates in MDA-MB-231 cells in vitro (Figure 3D). Then the Apc001OA-P4-VHL was named with Apc101 for further study.

To investigate whether Apc101 could induce the sclerostin/Apc101/VHL ternary complex, computational modeling¹⁹ and surface plasmon resonance (SPR) assay²⁰ were performed. The computational modeling data showed that Apc101 could bind both sclerostin and VHL to induce a sclerostin/Apc101/VHL ternary complex (Figure S10). Consistently, the SPR data demonstrated that Apc101 could bind both sclerostin and VHL to induce a sclerostin/Apc101/VHL ternary complex (KD = 6.31 nM) (Figure 3E). Moreover, confocal microscopy data showed that the Cy3 fluorescence signal was strong in cells treated with Cy3-labeled Apc101 rather than that in cells treated with Cy3-labeled scramble (Figures 3F and S11C). The western blot data showed that the intracellular sclerostin protein level was decreased with increasing concentration of Apc101 in TNBC cells (DC₅₀ = 0.53 μM for MDA-MB-231 cells, DC₅₀ = 0.50 μM for 4T1 cells) (Figures 3G, 3H, S11D, and S11E). Furthermore, the western blot data showed that intracellular sclerostin protein level was decreased from 3 h to a minimum of 42% at 6 h, and subsequently rebounded to 48% at 24 h in MDA-MB-231 cells treated with Apc101 (Figure 3I). The western blot data showed that intracellular sclerostin protein level was decreased from 3 h to a minimum of 42% at 6 h, and subsequently rebounded to 55% at 24 h in 4T1 cells treated with Apc101 (Figure S11F). To investigate the intracellular protein degradation selectivity of Apc101, we treated TNBC cells with Apc101 and PBS. The western blot data showed lower intracellular sclerostin level in Apc101 compared with the PBS group. Meanwhile, no difference was observed in the level of sclerostin domain containing-1 protein (SOSTDC1), sclerostin closest structural homolog, between Apc101 and PBS groups (Figure S12), indicating the high degradation selectivity of Apc101 for intracellular sclerostin.

To investigate the degrading mechanism of Apc101, we designed a negative control Apc101ˆ (with mutation in VHL E3 ligand to block the interaction between VHL E3 ligand and E3 ligase).²¹ The western blot data showed similar intracellular sclerostin protein level between Apc101ˆ and PBS groups (Figures 3J and S11G). Moreover, pretreatment with ubiquitination (MLN4924) or proteasome (MG132) inhibitors abolished Apc101-mediated degradation of intracellular sclerostin, as confirmed by western blot (Figures 3J and S11G), indicating that Apc101 could degrade intracellular sclerostin via VHL-dependent proteasome pathway.

To investigate the effect of Apc101 on TNBC cells in vitro, colony formation and transwell assays were performed on the above cells. Statistical data showed that colony formation and migration rates were significantly lower in the Apc101 group than in the PBS group both in MDA-MB-231 (Figures 3K and 3L) and 4T1 cells (Figures S11H and S11I). In addition, no significant differences were observed in colony formation and migration rates between the Apc101ˆ and PBS groups both in MDA-MB-231 (Figures 3K and 3L) and 4T1 cells (Figures S11H and S11I).

Taken together, the above genetic and pharmacological evidence consistently suggests the crucial oncogenic role of intracellular sclerostin in TNBC in vitro.

To investigate the effect of Apc101 on Wnt signaling in TNBC cells in vitro, the protein level of Wnt signaling marker β-catenin was compared between the PBS and Apc101 groups in TNBC cells using western blot analysis. Comparable β-catenin levels were observed between the PBS and Apc101 groups (Figure S13). Extracellular sclerostin is a Wnt signaling antagonist that binds to LRP5/6 on the cell membrane to inhibit bone formation.⁶ Interestingly, our data showed that intracellular sclerostin degradation by Apc101 did not affect Wnt signaling pathway in TNBC cells. This indicates that the promoting effects of intracellular sclerostin on TNBC proliferation and migration were independent of the Wnt signaling pathway.

The effect of Apc101 on tumor progression and metastasis in TNBC cell-derived mouse models

To investigate the effect of Apc101 on TNBC progression in vivo, MDA-MB-231 cell-inoculated orthotopic mice were treated with PBS, Taxel, Scl-Ab, and Apc101. The treatment schedule is shown in Figure 4A. The tumor images showed that the size of the primary tumors was smaller in the Apc101 group than in the PBS, Taxel, and Scl-Ab groups (Figure 4B). Tumor volume measurement data showed that the volume of primary tumors was significantly smaller in the Apc101 group than in the PBS, Taxel, and Scl-Ab groups (Figure 4C, left). The tumor weight measurement data showed that the weight of primary tumors was significantly lighter in the Apc101 group than in the PBS and Scl-Ab groups (Figure 4C, right). Moreover, no significant body weight loss was observed in any of the treatments (Figure 4D). Consistent findings were observed in 4T1 cell-inoculated orthotopic mice (Figures S15A–S15C). This indicates that Apc101 significantly inhibited tumor progression in TNBC cell-derived orthotopic mouse models.

Figure 4.

The effect of Apc101 on tumor progression and metastasis in MDA-MB-231 cell-inoculated orthotopic and metastatic mouse models

(A) A schematic diagram of treatment in MDA-MB-231 cell-inoculated orthotopic mouse model and metastatic mouse model.

(B) Representative images of primary tumor among PBS, Taxel, Scl-Ab, and Apc101 groups in MDA-MB-231 cell-inoculated orthotopic mouse model (n = 5 for each group).

(C and D) Statistics for comparing tumor growth rate and tumor weight (C) as well as body weight (D) among PBS, Taxel, Scl-Ab, and Apc101 groups in MDA-MB-231 cell-inoculated orthotopic mouse model (n = 5 for each group).

(E) Statistics for comparing lung metastatic nodule number among PBS, Taxel, Scl-Ab, and Apc101 groups in MDA-MB-231 cell-inoculated metastatic mouse model (n = 5 for each group).

Data are presented as mean ± SD. For in vivo studies, each spot represents one subject. For comparing tumor growth rate and body weight (C and D), p value was determined by two-way ANOVA with Dunnett post hoc test. For comparing tumor weight and number of lung metastatic nodules (C and E), p value was determined by one-way ANOVA with Dunnett post hoc test. NT, no tumor. ∗p < 0.05, ∗∗∗∗p < 0.0001.

To investigate the effect of Apc101 on TNBC metastasis in vivo, MDA-MB-231 cell-inoculated metastatic mice were treated with PBS, Taxel, Scl-Ab, and Apc101. The treatment schedule is shown in Figure 4A. Statistical data showed that the number of lung metastatic nodules was significantly fewer in the Apc101 group compared to the PBS and Scl-Ab groups (Figure 4E). Consistent findings were observed in 4T1 cell-inoculated orthotopic metastasis mice (Figure S15D). This indicates that Apc101 significantly inhibited lung metastasis in TNBC cell-derived metastatic mouse models.

To investigate the tissue distribution of Apc101, MDA-MB-231 cell-inoculated orthotopic mice were treated with PBS, Cy3-labeled Apc101, and Cy3-labeled Scl-Ab. The ex vivo fluorescence images demonstrated a strong Cy3 fluorescence signal of Apc101 in tumor and kidney tissues and a low Cy3 fluorescence signal of Apc101 in liver tissue (Figure S14). Notably, the data showed that the fluorescence intensity in tumor tissues was higher in tumor-bearing mice treated with Cy3-labeled Apc101 than that in mice treated with Cy3-labeled Scl-Ab (Figure S14).

Taken together, our findings indicate that Apc101 significantly inhibited tumor progression and lung metastasis in TNBC cell-derived mouse models, highlighting the vital role of intracellular sclerostin in TNBC.

The effect of Apc101 on tumor progression in TNBC PDX mouse models

To investigate the effect of Apc101 on tumor progression in the TNBC PDX mouse model, sclerostin-positive (H-score > 50) and sclerostin-negative (H-score < 50) clinical TNBC tissues were transplanted into mice. The treatment schedule is shown in Figure S16A. In the sclerostin-positive TNBC PDX mice, the tumor volume measurement data showed that the volume of primary tumors was significantly smaller in the Apc101 group than in the PBS group (Figure S16B). In contrast, in the sclerostin-negative TNBC PDX mice, no significant difference in the tumor volume was observed between the PBS and Apc101 groups (Figure S16C), indicating that the inhibitory effect of Apc101 on tumor progression was dependent on sclerostin expression in TNBC tissues. Consistently, our in vitro studies showed that Apc101 exhibited greater inhibitory effects on cell proliferation and migration in TNBC cells with relatively higher sclerostin expression than in those with relatively lower sclerostin expression (Figure S17). Taken together, these data indicate that the inhibitory effects of Apc101 on TNBC proliferation and migration are dependent on the expression of sclerostin.

We then established a PDX model using tumor tissue from a TNBC patient with sclerostin positivity (H-score > 50) to evaluate the anti-tumor effect of Apc101. The treatment schedule is shown in Figure 5A. The tumor images showed that the size of the primary tumors was smaller in the Apc101 (50 mg/kg) group than in the PBS, paclitaxel, and Apc101 (25 mg/kg) groups (Figure 5B). Tumor volume measurement data showed that the volume of primary tumors was significantly smaller in the Apc101 (50 mg/kg) group than in the PBS and paclitaxel groups (Figure 5C, left). Tumor growth inhibition analysis further confirmed the superior efficacy of Apc101 (50 mg/kg) compared with paclitaxel and Apc101 (25 mg/kg) (Figure 5C, right). Consistently, tumor weight was significantly lower in the Apc101 (50 mg/kg) group than in the PBS and Apc101 (25 mg/kg) groups (Figure 5D). Notably, statistical data showed that sclerostin protein expression was significantly lower in the Apc101 (50 mg/kg) group than in the PBS group by IHC assay (Figure 5E), suggesting good sclerostin degrading-activity of Apc101 in vivo. These results demonstrated that Apc101 significantly inhibited tumor progression in a dose-dependent manner in the sclerostin-positive TNBC PDX model, suggesting its strong therapeutic potential.

Figure 5.

The effect of Apc101 on tumor progression in sclerostin-positive TNBC PDX mouse model

(A) A schematic diagram of treatment in a sclerostin-positive TNBC PDX mouse model.

(B) Representative images of primary tumor among PBS, paclitaxel (5 mg/kg), Apc101 (25 mg/kg), and Apc101 (50 mg/kg) groups in a sclerostin-positive TNBC PDX mouse model (n = 5 for each group).

(C and D) Statistics for comparing tumor growth rate and TGI (C) as well as tumor weight (D) among PBS, paclitaxel (5 mg/kg), Apc101 (25 mg/kg), and Apc101 (50 mg/kg) groups in a sclerostin-positive TNBC PDX mouse model (n = 5 for each group).

(E) Representative images and statistics for comparing sclerostin H-score between PBS and Apc101 (50 mg/kg) groups. Scale bars, 50 μm.

Data are presented as mean ± SD. For in vivo studies, each spot represents one subject. For comparing tumor growth rate (C), p value was determined by two-way ANOVA with Dunnett post hoc test. For comparing tumor weight (D), p value was determined by one-way ANOVA with Dunnett post hoc test. For comparing sclerostin H-score (E), p value was determined by unpaired t test. TGI, tumor growth inhibition; s.c, subcutaneous; i.p, intraperitoneal. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Intracellular sclerostin and caprin1 mutually participated in interacting with and stabilizing CDK1 and Cyclin B1 mRNAs as well as in promoting TNBC proliferation and migration in vitro

The above findings reveal that intracellular sclerostin plays an important role in TNBC. However, the mechanism through which intracellular sclerostin works remains unclear.

In the pull-down assay, we identified cell cycle-associated protein 1 (caprin1) as an interactive sclerostin protein in MDA-MB-231 cells (Figure 6A). Caprin1, an RNA-binding oncoprotein, has been reported to stabilize STAT1 mRNA¹¹ and to promote tumor progression and metastasis.¹² Our in vitro studies showed that 231-SOST ^KD induced cell G2/M phase arrest in MDA-MB-231 cells (Figure S18). Furthermore, when compared with 231-SOST ^WT, 231-SOST ^KD exhibited lower levels of G2/M phase-associated mRNAs and higher degradation rates of CDK1 and Cyclin B1 mRNAs (Figures 6B and 6C), which are G2/M phase-associated mRNAs, to play important roles in promoting tumor progression and metastasis, respectively.^22,23 Moreover, when compared with 231-CAPRIN1 ^WT, 231-CAPRIN1 ^KD also exhibited lower levels of G2/M phase-associated mRNAs and higher degradation rates of CDK1 and Cyclin B1 mRNAs (Figures 6G and 6H). Consistent findings were also observed in 4T1 cells (Figure S19A, S19B, S19E, and S19F). It indicates that both intracellular sclerostin and caprin1 participate in maintaining CDK1 and Cyclin B1 mRNA stability in vitro.

Figure 6.

Intracellular sclerostin and caprin1 could mutually stabilize CDK1 mRNA and Cyclin B1 mRNA as well as promote TNBC proliferation and migration in MDA-MB-231 cells in vitro

(A) Identification of interactive proteins for sclerostin by pull-down assay.

(B) Statistics for comparing relative G2/M phase-associated mRNA levels between small interfering RNA (siRNA) negative control (NC) treatment and siRNA SOST treatment in 231-CAPRIN1^WT cells by RT-qPCR assay (left). Statistics for comparing relative CDK1 and Cyclin B1 mRNA levels between siRNA NC treatment and siRNA SOST treatment in 231-CAPRIN1^KD cells by RT-qPCR assay (right).

(C) Statistics for comparing degradation rates of CDK1 mRNA and Cyclin B1 mRNA between siRNA NC treatment and siRNA SOST treatment in 231-CAPRIN1^WT (upper) and 231-CAPRIN1^KD (lower) cells by RT-qPCR assay, respectively.

(D) Statistics for comparing relative levels of CDK1 mRNA and Cyclin B1 mRNA bound by intracellular sclerostin between 231-CAPRIN1^WT and 231-CAPRIN1^KD cells by RNA immunoprecipitation (RIP) assay.

(E and F) Statistics for comparing cell growth rate (E) and number of migrated cells (F) between siRNA NC treatment and siRNA SOST treatment in 231-CAPRIN1^WT and 231-CAPRIN1^KD cells, respectively.

(G) Statistics for comparing relative G2/M phase-associated mRNA levels between siRNA NC treatment and siRNA CAPRIN1 treatment in 231-SOST^WT cells by RT-qPCR assay (left). Statistics for comparing relative CDK1 and Cyclin B1 mRNA levels between siRNA NC treatment and siRNA CAPRIN1 treatment in 231-SOST^KD cells by RT-qPCR assay (right).

(H) Statistics for comparing degradation rates of CDK1 mRNA and Cyclin B1 mRNA between siRNA NC treatment and siRNA CAPRIN1 treatment in 231-SOST^WT (upper) and 231-SOST^KD (lower) cells by RT-qPCR assay, respectively.

(I) Statistics for comparing relative levels of CDK1 mRNA and Cyclin B1 mRNA bound by caprin1 between 231-SOST^WT and 231-SOST^KD cells by RIP assay.

(J and K) Statistics for comparing cell growth rate (J) and number of migrated cells (K) between siRNA NC treatment and siRNA CAPRIN1 treatment in 231-SOST^WT and 231-SOST^KD cells, respectively. For in vitro studies, error bars represent three independent experiments.

For comparing relative mRNA levels and number of migration cells (B, D, F, G, I, and K), p value was determined by one-way ANOVA with Dunnett post hoc test. For comparing degradation rates of mRNAs and cell growth rate (C, E, H, and J), p value was determined by two-way ANOVA with Dunnett post hoc test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

On one hand, our in vitro studies showed that 231-SOST ^WT CAPRIN1 ^WT cells exhibited higher levels of CDK1 and Cyclin B1 mRNAs and lower degradation rates of CDK1 and Cyclin B1 mRNAs compared with 231-SOST ^KD CAPRIN1 ^WT cells, whereas there were no differences in the levels and the degradation rates of CDK1 and Cyclin B1 mRNAs between 231-SOST ^WT CAPRIN1 ^KD and 231-SOST ^KD CAPRIN1 ^KD cells (Figures 6B and 6C). Consistent findings were also observed in 4T1 cells (Figures S19A and S19B). This indicates that the promoting effect of intracellular sclerostin on CDK1 and Cyclin B1 mRNA stability was dependent on caprin1. Furthermore, in our RNA immunoprecipitation (RIP) assay, we found that intracellular sclerostin could interact with CDK1 and Cyclin B1 mRNAs as an RNA-binding protein. However, lower levels of CDK1 and Cyclin B1 mRNAs bound to intracellular sclerostin were observed in 231-CAPRIN1 ^KD than in 231-CAPRIN1 ^WT cells, indicating that the interaction between intracellular sclerostin and CDK1 mRNA or Cyclin B1 mRNA was dependent on caprin1 in TNBC cells (Figure 6D). Moreover, our in vitro studies showed that 231-SOST ^WT CAPRIN1 ^WT cells exhibited higher cell growth and migration rates than 231-SOST ^KD CAPRIN1 ^WT cells, whereas there were no differences in the cell growth and migration rates between 231-SOST ^WT CAPRIN1 ^KD and 231-SOST ^KD CAPRIN1 ^KD cells (Figures 6E and 6F). Consistent findings were also observed in 4T1 cells (Figures S19C and S19D). This indicates that the promoting effects of intracellular sclerostin on TNBC proliferation and migration were dependent on caprin1.

On the other hand, our in vitro studies showed that 231-CAPRIN1 ^WT SOST ^WT cells exhibited higher levels of CDK1 and Cyclin B1 mRNAs and lower degradation rates of CDK1 and Cyclin B1 mRNAs compared with 231-CAPRIN1 ^KD SOST ^WT cells, whereas there were no differences in the levels and the degradation rates of CDK1 and Cyclin B1 mRNAs between 231-CAPRIN1 ^WT SOST ^KD and 231-CAPRIN1 ^KD SOST ^KD cells (Figures 6G and 6H). This indicates that the promoting effect of caprin1 on CDK1 and Cyclin B1 mRNA stability was dependent on intracellular sclerostin. Consistent findings were also observed in 4T1 cells (Figures S19E and S19F). In addition, lower levels of CDK1 and Cyclin B1 mRNAs bound by caprin1 were observed in 231-SOST ^KD than in 231-SOST ^WT cells, indicating that the interaction between caprin1 and CDK1 mRNA or Cyclin B1 mRNA was also dependent on intracellular sclerostin in TNBC cells (Figure 6I). Moreover, our in vitro studies showed that 231-CAPRIN1 ^WT SOST ^WT cells exhibited higher cell growth and migration rates than 231-CAPRIN1 ^KD SOST ^WT cells, whereas there were no differences in the cell growth and migration rates between 231-CAPRIN1 ^WT SOST ^KD and 231-CAPRIN1 ^KD SOST ^KD cells (Figures 6J and 6K). Consistent findings were also observed in 4T1 cells (Figures S19G and S19H). This indicates that the promoting effects of caprin1 on TNBC proliferation and migration were dependent on intracellular sclerostin.

Collectively, the above evidence indicates that intracellular sclerostin and caprin1 mutually participated in interacting with and stabilizing CDK1 and Cyclin B1 mRNAs, as well as promoting TNBC proliferation and migration in vitro.

Intracellular sclerostin-caprin1 interaction was required by intracellular sclerostin to promote TNBC proliferation and migration in vitro

Then, CAPRIN1 plasmids encoding CAPRIN1 WT and CAPRIN1 truncations were constructed to determine the interactive domains of caprin1 with sclerostin (Figure S20A). In the pull-down assay, it was found that the binding between sclerostin and CAPRIN1-T1/T2/T3/T4/T5 truncations still existed, whereas there was no binding between sclerostin and CAPRIN1-T6/T7 truncations (Figure S20B), indicating that the amino acids 249–290 of caprin1 were critical for the interaction between caprin1 and sclerostin. To further identify the residues responsible for the binding of caprin1 to sclerostin, 14 three-site-directed muteins of caprin1 were introduced into the CAPRIN1 WT plasmid (Table S1). In the pull-down assay, our in vitro data showed that the caprin1 mutein band intensity was lower in CAPRIN1-m1/m3/m8/m12/m14 than in the CAPRIN1 WT (Figure 7A). This suggests that 249T, 250H, 251N, 255G, 256L, 257C, 270D, 271Q, 272V, 282E, 283Y, 284T, 288E, 289V, and 290E could be the key binding sites of caprin1 for sclerostin. Additionally, we utilized Alphafold 3 to showcase the potential interaction modes of these two proteins, as illustrated in Figure 7B. Integrative analysis combining pull-down assay data with residue distance mapping demonstrated robust binding interfaces between the 251–259 residues of caprin1 and sclerostin (Figure 7B, lower). These findings provide a structural foundation for further investigation of the potential mechanisms.

Figure 7.

Blockade of intracellular sclerostin-caprin1 interaction could attenuate the promoting effects of intracellular sclerostin on the stability of CDK1 and Cyclin B1 mRNAs as well as TNBC proliferation and migration in MDA-MB-231 cells in vitro

(A) Binding analysis for the interaction between sclerostin and caprin1 muteins by pull-down assay.

(B) Predicted complex structure of sclerostin and caprin1 proteins using Alphafold 3 (left). Close-up view of the predicted binding interface, with key interacting residues labeled (upper right). Heatmap representation of residue interaction intensities between sclerostin and caprin1 residues 251–259 (lower right).

(C and D) Statistics for comparing cell growth rate (C) and relative migration % (D) between CAPRIN1 WT overexpression and CAPRIN1m overexpression in 231-SOST^WT cells. Scale bars, 200 μm.

(E) 231-SOST^WT cells were transfected with the following two constructs: (1) FLAG-ΔSP-hSOST plasmid and CAPRIN1 WT plasmid and (2) FLAG-ΔSP-hSOST plasmid and CAPRIN1m plasmid. RIP assay using anti-FLAG antibody was performed to compare the relative levels of CDK1 mRNA and Cyclin B1 mRNA bound by intracellular sclerostin between the two conditions (left). RT-qPCR assay was performed to compare the relative levels of CDK1 mRNA and Cyclin B1 mRNA between the two conditions (right).

(F) A schematic diagram for the role of intracellular sclerostin-caprin1 interaction in TNBC.

Data are presented as mean ± SD. For in vitro studies, error bars represent three independent experiments. For comparing cell growth rate (C), p value was determined by two-way ANOVA with Bonferroni post hoc test. For comparing relative migration and mRNA levels (D and E), p value was determined by unpaired t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

In 231-SOST ^KO cells, our in vitro studies showed that there were no differences in the cell growth and migration rates between the CAPRIN1 WT and CAPRIN1-m1/m3/m8/m14 groups (Figure S21), indicating that CAPRIN1-m1/m3/m8/m14 had no effects on TNBC proliferation and migration in the absence of sclerostin. However, in 231-SOST ^WT cells, our in vitro studies showed that the cell growth and migration rates were significantly lower in the CAPRIN1-m3 (G255A&L256A&C257A, named CAPRIN1m) group than in the CAPRIN1 WT (named CAPRIN1 WT) group (Figures 7C and 7D). To investigate whether intracellular sclerostin-caprin1 interaction could be required by intracellular sclerostin to interact with and stabilize CDK1 and Cyclin B1 mRNAs, RIP and quantitative reverse-transcription PCR (RT-qPCR) assays were conducted on 231-SOST ^WT cells overexpressing CAPRIN1 WT and CAPRIN1m, respectively. In the RIP assay, our data demonstrated that the levels of CDK1 and Cyclin B1 mRNAs bound to intracellular sclerostin were lower in the CAPRIN1m group than in the CAPRIN1 WT group, indicating that the intracellular sclerostin-caprin1 interaction could be required by intracellular sclerostin to interact with CDK1 and Cyclin B1 mRNAs (Figure 7E, left). In the RT-qPCR assay, our in vitro studies consistently showed that the levels of CDK1 and Cyclin B1 mRNAs were lower in the CAPRIN1m group than in the CAPRIN1 WT group, indicating that the intracellular sclerostin-caprin1 interaction could be required by intracellular sclerostin to stabilize CDK1 and Cyclin B1 mRNAs (Figure 7E, right). Collectively, the above data suggest that intracellular sclerostin-caprin1 interaction is required by intracellular sclerostin to stabilize CDK1 and Cyclin B1 mRNAs and to promote TNBC proliferation and migration in vitro.

In summary, intracellular sclerostin cooperatively interacts with caprin1 to stabilize CDK1 and Cyclin B1 mRNAs and to promote TNBC progression and metastasis (Figure 7F). Together, our findings elucidate a previously unrecognized role of intracellular sclerostin in TNBC, suggesting that targeting intracellular sclerostin may represent a potent therapeutic strategy for patients who currently lack effective treatment options.

Discussion

This study identifies intracellular sclerostin as an oncogenic driver in TNBC, marking a significant departure from its established role in regulating bone formation.^6,24 A previous study reported that SOST mRNA was detected in 56% of the clinical TNBC tissues.⁸ However, neither sclerostin protein expression level nor its role was investigated further. In our clinical TNBC biobank, 60% of the tumor tissues exhibited sclerostin protein expression (H-score > 50), which was associated with the elevated tumor proliferation marker, indicating the potential of sclerostin as a biomarker for disease progression. Furthermore, Apc101 treatment significantly suppressed tumor progression in both TNBC cell-derived xenograft and sclerostin-positive PDX models, but not in the sclerostin-negative TNBC PDX model, emphasizing the importance of biomarker-driven approaches. This indicates that sclerostin could also be developed as a therapeutic target for a precision medicine-based therapeutic strategy, ultimately benefiting the majority of TNBC patients.

The rationale for intracrine regulation, rather than autocrine signaling in cancer cells, warrants further exploration. We propose that tumors might optimize survival strategies by prioritizing intracellular pathway activation, which reduces their dependence on the extracellular microenvironment and minimizes resource allocation to secreted factors. This paradigm aligns with emerging evidence that underscores the multifunctional roles of canonical secretory or membrane proteins in cancer biology. For instance, S100A8/A9, which is classically characterized as a secreted cytokine, has also been reported as a nuclear transcriptional coactivator in breast cancer cells.²⁵ Similarly, intracellular PD-L1, beyond its well-established role as an extracellular immune checkpoint regulator, has been identified as a potential therapeutic target for enhancing radiotherapy and chemotherapy efficacy in cancer treatment.²⁶ These findings suggest that the intracellular activation of survival pathways might represent a more efficient and tightly regulated mechanism for sustaining cancer cell proliferation and migration. Furthermore, our findings indicate that systematic exploration of the intracellular functions of secretory proteins, especially those not conventionally linked to cancer, could reveal unrecognized oncogenic mechanisms and potential therapeutic targets.

Although it was well known that sclerostin was a Wnt signaling antagonist,^6,24 our study challenged this conventional role in TNBC. We identified caprin1 as a binding partner of intracellular sclerostin in TNBC cells. It was reported that caprin1 participated in protecting STAT1 mRNA from degradation.¹¹ In this study, we identified another binding partner, intracellular sclerostin, for caprin1 to stabilize mRNAs, thereby advancing our understanding of how caprin1 acts as an RNA-binding protein to stabilize mRNAs. Further studies will be required to determine whether intracellular sclerostin-caprin1 interaction could influence the stability of other oncogenic mRNAs. This study bridges the understanding gap of how intracellular sclerostin promotes TNBC progression and metastasis. This suggests that specific blockade of the intracellular sclerostin-caprin1 interaction could be developed as a potential therapeutic strategy for TNBC.

Collectively, this study reveals an unrecognized oncogenic function of intracellular sclerostin in TNBC and indicates that targeting it might represent a promising therapeutic strategy. Furthermore, our findings highlight the need for systematic exploration of the intracellular functions of secretory proteins, particularly those not conventionally linked to cancer, to uncover unrecognized oncogenic mechanisms and therapeutic targets.

Limitations of the study

While this study systematically elucidates how intracellular sclerostin drives TNBC progression and metastasis, certain limitations warrant further investigation. First, to enhance the translational potential of Apc101, its anti-tumor efficacy should be validated in sclerostin-positive/Trop2-low PDX models, a patient population with limited therapeutic options. Second, the specific molecular basis for how intracellular sclerostin stabilizes CDK1 and Cyclin B1 mRNAs remains to be further investigation. Last, a more comprehensive understanding of its functional landscape via RIP sequencing is warranted to uncover its global impact on mRNA stability and gene regulatory networks.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Ge Zhang (zhangge@hkbu.edu.hk).

Materials availability

All reagents generated in this study are available upon request from the lead contact with a completed materials transfer agreement.

Data and code availability

• •All data generated or analyzed during this study are included in this manuscript (and its supplemental information files). This paper also analyzes existing, publicly available data (deposited data of STAR Methods), which is available at Zenodo (https://doi.org/10.5281/zenodo.18752090).
• •This study does not report original code.
• •Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

This study was supported by the National Key R&D Program of China (no. 2018YFA0800804); Hong Kong General Research Fund from the Research Grants Council of the Hong Kong Special Administrative Region, China (project nos. 12102120, 14103121, 14103420, and 14109721); Theme-Based Research Scheme from the Research Grants Council of the Hong Kong Special Administrative Region, China (project no. T12-201/20-R); Basic and Applied Basic Research Fund from Department of Science and Technology of Guangdong Province (project no. 2019B1515120089); Inter-Institutional Collaborative Research Scheme from Hong Kong Baptist University (project no. RC-ICRS/19–20/01); the direct grant of The Chinese University of Hong Kong (project no. 4054660); Youth's Project of Guangdong Basic and Applied Basic Research Fund (GDSTC no. 2022A1515110044); the Innovation and Technology Support Programme (Innovation and Technology Commission, ITS/523/24FP); and the Young Scientists Fund (the National Natural Science Foundation of China, 22507110). We also thank Hilda Cheung and David Wong for their technical support.

Author contributions

G.Z., Q.C., B.-T.Z., F.L., Y.M., F.-L.Y., and A.L. supervised the project. M.S., H.L., Z.C., S.Q., H.J., and X.Y. performed the major research and wrote the manuscript with equal contributions. S.D., Y.T., Y.W., P.W., Y.Z., and X.T. provided technical support. Y.Y., L.W., W.K., J.T., D.M., and Y.H. provided professional expertise.

Declaration of interests

Y.H. was employed by Aptacure Therapeutics Limited.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit polyclonal anti-Sclerostin	Thermo Fisher Scientific	Cat# PA5-113315; RRID: AB_2868049
Rabbit polyclonal anti-Ki-67	Proteintech	Cat# 27309-1-AP; RRID: AB_2756525
Rabbit polyclonal anti-Sclerostin	Abcam	Cat# ab85799; RRID: AB_10859114
Rabbit polyclonal anti-SOSTDC1	Proteintech	Cat# 22213-1-AP; RRID: AB_3085688
Rabbit polyclonal anti-β-actin	Proteintech	Cat: 20536-1-AP; RRID: AB_10700003
Mouse monoclonal anti-His (H-3)	Santa Cruze Biotechnology	Cat# sc-8036; RRID: AB_627727
Rabbit polyclonal anti-Caprin-1	Abcam	Cat# ab244360; RRID: AB_2313773
Rabbit monoclonal anti-Flag (D6W5B)	Cell Signaling Technology	Cat# 14793; RRID: AB_2572291
Goat polyclonal secondary Anti-rabbit IgG, HRP-linked Antibody	Cell Signaling Technology	Cat# 7074; RRID: AB_2099233
Horse polyclonal secondary Anti-mouse IgG, HRP-linked Antibody	Cell Signaling Technology	Cat# 7076; RRID: AB_330924
Biological samples
Tumor tissues from breast cancer patients	Guangdong Provincial Hospital of Chinese Medicine, Guangzhou, China	N/A
Tumor tissues from breast cancer patients	Affiliated Hospital of Jiangnan University	N/A
Patient-derived xenografts (PDX)	Guangdong Provincial Hospital of Chinese Medicine, Guangzhou, China	N/A
Chemicals, peptides, and recombinant proteins
Human Sclerostin	Sino Biological	Cat# 10593-H07H-B
Mouse Sclerostin	R&D Systems	Cat# 1589-ST-025/CF
4% Paraformaldehyde	Beyotime	Cat# P0099
Crystal violet	Beyotime	Cat# C0121
DAPI Mounting Medium	Abcam	Cat# ab104139
TRIzol reagent	Thermo Fisher Scientific	Cat# 15596026
D-luciferin	Sigma-Aldrich	Cat# 50227
2x Rapid Taq Master Mix	Vazyme	Cat# P222
Actinomycin D	Thermo Fisher Scientific	Cat# 11805017
N3-PEG4-CH2COONHS	ACHEMBLOCK	Cat# V156175
(S, R, S)-AHPC-PEG4-N3	MedChemExpress	Cat# HY-103601
Thalidomide-O-PEG4-azide	MedChemExpress	Cat# HY-140844
Nutlin-C1-amido-PEG4-C2-N3	MedChemExpress	Cat# HY-128832
N3-PEG2-C2-NHS ester	leyan	Cat# 1175728
Azido-PEG6-NHS ester	BROADPHARM	Cat# BP-21506
Azido-PEG8-NHS ester	BROADPHARM	Cat# BP-21606
Azido-PEG10-NHS ester	BROADPHARM	Cat# BP-23998
(S, R, S)-AHPC	MedChemExpress	Cat# HY-125845
NHS-Fluorescein	Thermo Scientific	Cat# 46410
Cy3-NHS	leyan	Cat# LY-JK-9389050
Copper (II) sulfate pentahydrate	Sigma-AIdrich	Cat# 469130
Tris(2-carboxyethyl) phosphine hydrochloride	MACKLIN	Cat# T819166
N,N-Diisopropylethylamine	MACKLIN	Cat# N741860
Ultracel-10K	Merck Millipore	Cat# UFC501096
Sephadex G-25 DNA Grade	Cytiva	Cat# 17085302
MLN4924	MedChemExpress	Cat# HY-70062
MG132	MedChemExpress	Cat# HY-13259
(2S,4S)-1-((S)-2-Amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl) benzyl)pyrrolidine-2-carboxamide hydrochloride	bidepharm	Cat# BD01272772
VHL/ELOB/ELOC Complex	LifeSensors	Cat# UB332
TBS (20×)	Beyotime	Cat# ST663-500 mL
TWEEN 20	Sigma-AIdrich	Cat# P1379-500ML
Nonfat powdered milk	Beyotime	Cat# P0216-1500g
Blocking Buffer for Immunol Staining	Beyotime	Cat# P0260
Lipomaster 3000 Transfection Reagent	Vazyme	Cat# TL301-01
Critical commercial assays
His Protein Interaction Pull-Down Kit	Thermo Fisher Scientific	Cat# 21277
Silver Stain Kit	Thermo Fisher Scientific	Cat# 24612
High-Capacity RNA-to-cDNA Kit	Thermo Fisher Scientific	Cat# 4387406
RNA-Binding Protein Immunoprecipitation Kit	Sigma-Aldrich	Cat# 17-700
Immunochromogenic reagent GTVisionTM Ⅲ Detection System	Gene Tech	Cat# GK500710
Deposited data
Raw and analyzed data	This paper	GEO: GSE21653, GSE20685, GSE31448, GSE58812. Zenodo:https://doi.org/10.5281/zenodo.18752090
Experimental models: Cell lines
Human: MDA-MB-231	ATCC	HTB-26
Mouse: 4T1	ATCC	CRL-2539
Human: HCC1806	ATCC	CRL-2335
Experimental models: Organisms/strains
Mouse: BALB/cJGpt	GemPharmatech LLC.	N000020
Mouse: BALB/c-Nude mice	GemPharmatech LLC.	D000521
Mouse: BALB/cJGpt-Sost cas9-ko	GemPharmatech LLC.	N/A
Oligonucleotides
Primers for target genes, see Table S2	This paper	N/A
Recombinant DNA
Plasmid: pcDNA3.1-CAPRIN1-WT-3xHA	This paper	N/A
Plasmid: pcDNA3.1-CAPRIN1-606-3xHA	This paper	N/A
Plasmid: pcDNA3.1-CAPRIN1-380-3xHA	This paper	N/A
Plasmid: pcDNA3.1-CAPRIN1-351-3xHA	This paper	N/A
Plasmid: pcDNA3.1-CAPRIN1-321-3xHA	This paper	N/A
Plasmid: pcDNA3.1-CAPRIN1-291-3xHA	This paper	N/A
Plasmid: pcDNA3.1-CAPRIN1-248-3xHA	This paper	N/A
Plasmid: pcDNA3.1-CAPRIN1-100-3xHA	This paper	N/A
Plasmid: pcDNA3.1-CAPRIN1-1m-3xHA	This paper	N/A
Plasmid: pcDNA3.1-CAPRIN1-2m-3xHA	This paper	N/A
Plasmid: pcDNA3.1-CAPRIN1-3m-3xHA	This paper	N/A
Plasmid: pcDNA3.1-CAPRIN1-4m-3xHA	This paper	N/A
Plasmid: pcDNA3.1-CAPRIN1-5m-3xHA	This paper	N/A
Plasmid: pcDNA3.1-CAPRIN1-6m-3xHA	This paper	N/A
Plasmid: pcDNA3.1-CAPRIN1-7m-3xHA	This paper	N/A
Plasmid: pcDNA3.1-CAPRIN1-8m-3xHA	This paper	N/A
Plasmid: pcDNA3.1-CAPRIN1-9m-3xHA	This paper	N/A
Plasmid: pcDNA3.1-CAPRIN1-10m-3xHA	This paper	N/A
Plasmid: pcDNA3.1-CAPRIN1-11m-3xHA	This paper	N/A
Plasmid: pcDNA3.1-CAPRIN1-12m-3xHA	This paper	N/A
Plasmid: pcDNA3.1-CAPRIN1-13m-3xHA	This paper	N/A
Plasmid: pcDNA3.1-CAPRIN1-14m-3xHA	This paper	N/A
Plasmid: pcDNA3.1-hSOST-3xFlag	This paper	N/A
Plasmid: pcDNA3.1-mSost-3xFlag	This paper	N/A
Software and algorithms
GraphPad Prism (10.4.2)	GraphPad Software	https://www.graphpad.com/
QuPath (0.4.2)	Queens University Belfast; Ireland; United Kingdom	https://qupath.github.io/
Living Image software (4.5)	Caliper Life Sciences	https://www.perkinelmer.com/
BioRender	N/A	https://biorender.com/
ImageJ	National Institutes of Health	https://imagej.net/ij/
UCSF ChimeraX (1.10)	University of California, San Francisco	https://www.cgl.ucsf.edu/chimerax/
Flowjo (v10)	TreeStar	https://www.flowjo.com/solutions/flowjo/downloads
LAS X (3.1.1.15751)	Leica Microsystems	https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/

Experimental model and study participant details

Human samples

Written informed consent was obtained from all breast cancer patients participated in this study and the use of patients’ specimens was conducted with the approval of the Ethics Committee of Affiliated Hospital of Jiangnan University (Approval No. LS2023109) and the Guangdong Provincial Hospital of Chinese Medicine (Approval No. BE2024-182-01). A total of 293 tumor samples derived from Chinese female breast cancer patients were enrolled across three distinct cohorts. There was no influence of sex or gender on the results of our study. The TNBC cohort included 100 female patients across all stages, with a median age of 54.5 years (range: 28–88 years). The HER2-positive cohort included 94 female patients, with a median age of 53 years (range: 30–77 years). The luminal cohort included 99 female patients, with a median age of 51 years (range: 21–83 years). These samples were subjected to IHC analysis.

Cell lines and cell culture

MDA-MB-231 cell line (ATCC HTB-26) is a human TNBC cell line widely used in xenograft mouse model. It was cultured in DMEM (ATCC, Cat: 30–2002), supplemented with 10% fetal bovine serum (FBS), and 100 U/ml penicillin-streptomycin (PS).²⁷ The modified MDA-MB-231 cell lines with the mCherry reporter, 231-SOST ^WT and 231-SOST ^KO were maintained in the same complete medium as MDA-MB-231 cells. 4T1 cell line (ATCC CRL-2539) is a murine TNBC cell line widely used in syngeneic mouse model for TNBC spontaneous metastatic research. It was cultured in RPMI-1640 (ATCC, Cat: 30–2001) with 10% FBS and 100 U/ml PS.²⁸ The modified 4T1 cell lines with luciferase reporter, 4T1-sost ^WT and 4T1-sost ^KO, were maintained in the same complete medium as 4T1 cells. HCC1806 (ATCC CRL-2335) is another human TNBC cell line that was cultured in RPMI-1640 (ATCC, Cat: 30–2001) with 10% FBS and 100 U/ml PS. Cell lines were all cultured at 37°C with 5% CO₂ and routinely tested for mycoplasma contamination to ensure sterility.

Animal experiments

To establish MDA-MB-231 orthotopic mouse model, female athymic nude mice (6–8 weeks old) were anesthetized with 2.5% isoflurane and subcutaneously (s.c.) injected with 2 × 10⁶ cells suspended in 50 μL of a 1:1 Matrigel:PBS mixture into the abdominal mammary fat pad, respectively. Six weeks post-inoculation, the mice were euthanized, and the orthotopic tumors were harvested for photography. Tumor dimensions (length [L] and width [W]) were measured twice a week using calipers. Tumor volume (mm³) was determined using the following formula: “V = L × W²/2”. The tumor growth curve was plotted using GraphPad on the volume data monitored for evaluation of the orthotopic tumor progression.²⁹ To establish 4T1 orthotopic mouse model, female Balb/c mice (6–8 weeks old) were anesthetized with 2.5% isoflurane and subcutaneously (s.c.) injected with 1 × 10⁵ cells into the abdominal mammary gland area using a 50 μL single-cell suspension in PBS, respectively.³⁰ Three weeks post-inoculation, the mice were euthanized, and the orthotopic tumors were harvested for photography. To establish MDA-MB-231 metastasis mouse model, female athymic nude mice (6–8 weeks old) were anesthetized with 2.5% isoflurane and received an intracardiac (i.c.) injection of 2 × 10⁵ cells suspended in 100 μL ice-cold PBS into the left ventricle, respectively. Six weeks post-inoculation, the mice were euthanized to harvest lung tissues to quantify lung metastatic nodules.^31,32 To establish 4T1 metastasis mouse model, female Balb/c mice (6–8 weeks old) were anesthetized with 2.5% isoflurane and intravenously (i.v.) injected with 1 × 10⁵ cells using a 50 μL single-cell suspension in PBS, respectively.²⁸ Three weeks post-inoculation, the mice were euthanized to harvest the lung tissues for ex vivo bioluminescence imaging and quantification of lung metastatic nodules. To establish TNBC patient-derived xenograft (PDX) mouse model, fresh TNBC tissues were obtained from two treatment-naïve female patients: one with sclerostin positive TNBC tissue (H-score > 50) and the other with sclerostin negative TNBC tissue (H-score < 50). Tissue specimens from each patient were aseptically dissected into 3 × 3 × 3 mm³ fragments and orthotopically implanted into the mammary fat pads of female NCG mice (6–8 weeks old). When tumors reached 1–2 cm in diameter³ (first passage), they were excised and transplanted into a new cohort of NCG mice (second passage). This serial transplantation protocol was repeated to establish stable PDX models through third to fifth passages (Approval No. BE2022-204).³³ All animals were housed in pathogen-free containment with a 12-h light-dark cycle and ad libitum food and water. All cell-derived xenograft (CDX) animal experimental protocols in this study were approved by the Research Ethics Committee of the Hong Kong Baptist University. All patient-derived xenograft (PDX) animal experimental protocols in this study were approved by the Research Ethics Committee of the Guangdong Provincial Hospital of Chinese Medicine. All experiments for individual mice were concluded either when the total tumor volume exceeded 1,500 mm³ or in cases of ulcerated tumors.

Method details

Immunohistochemical (IHC) analysis

IHC analysis was conducted on paraffin-embedded breast cancer tissue sections using standardized protocols.²⁹ The procedure involved rehydration, endogenous peroxidase blocking with 3% hydrogen peroxide, and antigen retrieval using heat-induced epitope recovery in citrate buffer. After blocking (Beyotime, Cat: P0260), the sections were incubated overnight at 4°C with the primary antibodies against sclerostin (Thermo Fisher Scientific, Cat: PA5-113315; 1:500) or Ki-67 (Proteintech, Cat: 27309-1-AP; 1:500), followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (Gene Tech, Cat: GK500710). Signal detection was performed using diaminobenzidine (DAB) chromogen substrate, followed by hematoxylin nuclear counterstaining for 3 min. The slides were then visualized under a microscope. 3–5 random fields from each slide were quantified using QuPath software. For sclerostin IHC scoring, intensity thresholds were established to classify cells into the categories of negative (0), weak (1), moderate (2) or strongly positive (3) for sclerostin staining determined by mean cytoplasm DAB optical densities. The H-score was calculated by summing three times the percentage of cells that were strongly stained, two times the percentage of cells that were moderately stained, and one time the percentage of cells that were weakly stained, yielding values between 0 (no staining in cell cytoplasm) and 300 (strongly positive staining in all cell cytoplasm). An H-score > 50 was used as the threshold for sclerostin positivity.³⁴

Preparation of sclerostin aptamer-based PROTACs

In the plastic reaction tube, 5′-alkyne sclerostin aptamer Apc001OA or Cy3-labled Apc001OA (1.0 eq),¹⁸ azide linked-VHL/CRBN/MDM2 ligand ((S, R, S)-AHPC-PEG4-N3, MedChemExpress, Cat: HY-103601) (Thalidomide-O-PEG4-azide, MedChemExpress, Cat: HY-140844) (Nutlin-C1-amido-PEG4-C2-N3, MedChemExpress, Cat: HY-128832) or azide linked-VHL ligands with different linkers (100 eq), and Copper (II) sulfate pentahydrate (Sigma-AIdrich, Cat: 469130) (50 eq) were transferred. For the azide linked-VHL ligands with different linkers, which were generated by mixing the azide-linker-NHS esters (N3-PEG2-C2-NHS ester, leyan, Cat: 1175728) (N3-PEG4-CH2COONHS, ACHEMBLOCK, Cat: V156175) (Azido-PEG6-NHS ester, BROADPHARM, Cat: BP-21506) (Azido-PEG8-NHS ester, BROADPHARM, Cat: BP-215606) (Azido-PEG10-NHS ester, BROADPHARM, Cat: BP-23998) (1.0 eq), NH₂-VHL ligand ((S, R, S)-AHPC, MedChemExpress, Cat: HY-125845) ((2S,4S)-1-((S)-2-Amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl) benzyl)pyrrolidine-2-carboxamide hydrochloride, bidepharm, Cat: BD01272772) (1.5 eq), and N,N-Diisopropylethylamine (MACKLIN, Cat: N741860) (1.5 eq) in DMSO for 4 h, respectively. Then, NaHCO₃ (200mM) and CH₃CN/H₂O (V_CH3CN/V_H2O = 6%) were added to the mixture, followed by brief vortexing. Next, Tris(2-carboxyethyl) phosphine hydrochloride (MACKLIN, Cat: T819166) (20mM) was added. The reaction tube was then briefly purged by nitrogen gas and kept on shaker at 25°C for next 12 h. After completion of reaction, the reaction mixture was purified by Sephadex G-25 DNA Grade (Cytiva, Cat: 17085302) Columns. The crude products were further purified by HPLC using a gradient method with 0.05 M TEAA and acetonitrile as solvents. The appropriate fractions were collected and then confirmed by ESI-MS analysis.³⁵

Preparation of fluorescein or Cy3-labeled sclerostin antibody (Scl-Ab)

To label Scl-Ab with Fluorescein or Cy3, Scl-Ab (1 eq) was dissolved in PBS (pH 8.0) containing NHS-Fluorescein (Thermo Scientific, Cat: 46410) (15 eq) or Cy3-NHS (leyan, Cat: LY-JK-9389050) (15 eq). The mixture was then incubated at 25°C for 2 h in the dark.³⁶ The final solution was then filtered (cutoff: 10k Da) (Ultracel-10K, Merck Millipore, Cat: UFC501096) to remove the free NHS-Fluorescein and Cy3-NHS.

Ternary complex modeling

The crystal structures of sclerostin and VHL were obtained from PDB entries 2K8P and 4W9C, respectively. Aptamer structures were predicted using trRosettaRNA. Molecular docking was performed using HADDOCK 2.4 to generate protein-aptamer binary complexes, E3 ligand-E3 ligase complexes, and protein-protein complexes.¹⁹ Linker conformations were generated with RDKit, and the complete aptamer-based PROTAC structures were assembled in PyMOL by connecting the aptamer and E3 ligand components through the linker moieties. All structural visualizations were prepared using ChimeraX.

SPR assay

SPR experiments were performed on Biacore T200 at 25°C in a running buffer containing 1×PBS pH 7.4 and 0.005% Tween 20. The sclerostin was immobilized on a CM5 chip at 25°C, and the final surface density of sclerostin was approximately 1400 RU. Then Apc101 (4 μM) was injected to load sclerostin with Apc101. The VHL/ELOB/ELOC (LifeSensors, Cat: UB332) solutions (7.5–120 nM) were injected to a multi-cycle kinetic mode (association time 120 s, dissociation time 300 s, regeneration time 30 s, flow rate 30 μL/min). The equilibrium dissociation constant (KD) was obtained by kinetic fitting (1:1).²⁰

Colony formation assay

Single-cell suspensions of 231-SOST ^WT and 231-SOST ^KO cells were plated in 6-well plates with complete DMEM medium, respectively. Single-cell suspensions of 4T1-sost ^WT and 4T1-sost ^KO cells were plated in 6-well plates with complete RPMI-1640 medium, respectively. The indicated treatments were administered on the following day. Fresh medium containing the respective treatments was replaced every 48 h until visible colonies were formed. After 9–14 days of incubation, the cells were rinsed with PBS, fixed in 4% paraformaldehyde (PFA; Beyotime, Cat: P0099), and stained with crystal violet (Beyotime, Cat: C0121). Colony images were captured using an Epson office scanner in reflective mode, and colony quantification was performed using ImageJ software.³⁷ For statistical analysis, data were expressed as a percentage relative to the mean of the control group.³⁸

Transwell migration and invasion assay

Cell migration and invasion were assessed using transwell inserts (Corning, Cat: 353097) for migration assay and Matrigel-coated invasion chambers (Corning, Cat: 354480) for invasion assay, respectively. For MDA-MB-231 cells, 231-SOST ^WT and 231-SOST ^KO cells were collected in serum-free DMEM medium and seeded at 5 × 10⁴ cells per well into the top chambers (for migration or invasion assays). For 4T1 cells, 4T1-sost ^WT and 4T1-sost ^KO cells were collected in serum-free RPMI-1640 medium and seeded at 1 × 10⁵ cells per well into the top chambers (for migration or invasion assays). Complete medium was placed in the lower chamber to serve as the chemoattractant. After 20–24 h of migration/invasion, the cells were rinsed with PBS, fixed in 4% PFA (Beyotime, Cat: P0099), and stained with crystal violet (Beyotime, Cat: C0121). Non-migrated/invaded cells were mechanically removed from the membrane surface by cotton swab abrasion, while migrated/invaded cells on the basal side of the membrane were visualized using a Leica DMI3000 microscope. Quantification was performed by counting the cells in five random microscopic fields per membrane using ImageJ software.³⁹

Confocal microscopy imaging

To investigate the internalization of Scl-Ab, the cells were seeded in glass-bottom confocal dishes. The cells were then treated with 10 μg/mL Fluorescein-labeled IgG or Scl-Ab at 37°C for 3 h. Similarly, for Apc101 internalization, the cells were incubated overnight in confocal dishes and treated with 5 μg/mL (0.35μM) Cy3-labeled scramble control or Apc101 at 37°C for 3 h. Following treatment, the cells were washed with PBS, fixed with 4% PFA (Beyotime, Cat: P0099), and permeabilized with 0.1% Triton X-100 for 10 min. After additional PBS washes, the cells were mounted with DAPI-containing medium (Abcam, Cat: ab104139) and imaged using confocal microscopy.⁴⁰ Images were acquired using LAS X software and confocal microscopy.

Fluorescence imaging

For in vivo fluorescence live imaging, the mice were anesthetized with 2.5% isoflurane prior to live imaging. Fluorescence signals from 231-SOST ^WT and 231-SOST ^KO tumors (engineered with mCherry reporters) were captured using an IVIS Lumina XR imaging system. All images were acquired under standardized illumination settings. Fluorescence intensity was quantified using region of interest (ROI) analysis in Living Image 4.5 software.

Bioluminescence imaging

For in vivo bioluminescence live imaging, the mice were anesthetized with 2.5% isoflurane and intraperitoneally (i.p.) injected with 100 μL D-luciferin (30 mg/mL; Sigma-Aldrich, Cat: 50227) 10 min prior to live imaging. Bioluminescent signals from 4T1-sost ^WT and 4T1-sost ^KO tumors (engineered with luciferase reporters) were captured using an IVIS Lumina XR imaging system.⁴¹ For ex vivo bioluminescence live imaging, following anesthesia and D-luciferin injection as described above, mice were euthanized, and the lung tissues were immediately harvested in a 24-well plate with 150 μg/mL D-luciferin for imaging. All images were acquired under standardized illumination settings. Bioluminescence intensity was quantified using ROI analysis in Living Image 4.5 software.

Lung metastasis analysis

For quantification of lung metastatic nodules in mouse models, the lung tissues were rinsed with PBS to clear residual blood and subsequently fixed in 4% PFA (Beyotime, Cat: P0099). The lung tissues were then photographed. And the visible nodules in the lung tissues were counted. Following fixation, the lung tissues were paraffin-embedded and the largest cross-sectional plane of each intact lung was selected for analysis. Sequential 5μm thick paraffin sections were prepared and stained with hematoxylin and eosin (H&E) for histological evaluation. Whole-lung images were acquired using a Pannoramic MIDI slide scanner. Metastatic nodules were identified as dark purple lesions in H&E-stained sections and quantitatively assessed using QuPath software.⁴²

Genotyping

Sclerostin-deficient transgenic mice (SOST ^−/−) with full-length gene ablation were obtained from GemPharmatech Co., Ltd. Genotyping of the SOST ^−/− allele was performed by using DNA isolated from mouse tail biopsies. PCR amplification was carried out with the following primer pairs: KO forward primer 5′-TCAATAGTAGGGTGCTTGCACAGC-3′, KO reverse primer 5′ AGAACCACGTAGCCCAACATCAC-3′, WT forward primer 5′- TCCTAAGGATGAGGACACAGGTCAG-3′, and WT reverse primer 5′- CATGTCACATGCCACCTACAAACC-3’. Reactions utilized 2x Rapid Taq Master Mix (Vazyme, Cat: P222). Homozygous SOST ^−/− mice exhibited only a 523 bp KO amplicon, whereas no WT product was detected.²⁴

Western blot assay

To investigate the degrading mechanism of Apc101, MDA-MB-231 cells and 4T1 cells were pretreated with MLN4924 (1 μM) (MedChemExpress, Cat: HY-70062) and MG132 (1 μM) (MedChemExpress, Cat: HY-13259) for 1h, respectively. Then the protein samples were separated by 10% SDS-PAGE gel using a two-step electrophoresis protocol (80 V for 30 min, then 110 V for 60 min). Following separation, proteins were transferred to polyvinylidene difluoride (PVDF) Transfer membrane (Thermo Scientific, Cat: 88518) by electroblotting under the following conditions: 350 mA for 60 min. Membranes were then blocked with 5% (w/v) Nonfat powdered milk (Beyotime, Cat: P0216-1500g) in Tris-buffered saline (Beyotime, Cat: ST663-500 mL) containing 0.1% Tween 20 (Sigma-AIdrich, Cat: P1379-500ML). Primary antibody incubation was performed overnight at 4°C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system (Bio-Rad) according to the manufacturer’s protocol. All western blot images were processed using Image Lab software.⁴³

Pull-down assay

Pull-down assay was conducted according to the manufacturer’s protocol (Thermo Fisher Scientific, Cat: 21277). Briefly, His-tagged recombinant human sclerostin protein (Sino Biological, Cat: 10593-H07H-B) was incubated with pre-equilibrated HisPur Cobalt Resin on a rotary shaker at 4°C for 2 h to immobilize the bait protein (His-tagged sclerostin) onto the resin. Unbound proteins were then removed by washing the resin five times with wash buffer. Lysates from control or plasmid-transfected cells via Lipofectamine 3000 (Vazyme, Cat: TL301-01) were then prepared and incubated overnight at 4°C with protein-resin complexes, followed by five additional washes with wash buffer. Subsequently, bound proteins were eluted and analyzed via SDS-PAGE for subsequent mass spectrometry (MS) or western blot to identify the proteins interacting with sclerostin.⁴⁴

Silver staining

To identify the interactive proteins for sclerostin in TNBC, silver staining was conducted for SDS-PAGE gel after the pull-down assay (Thermo Fisher Scientific, Cat: 24612). Distinct protein bands of interest were manually excised from the gel for subsequent MS analysis.

Flow cytometry analysis

Following siRNA-mediated gene silencing, the cells were harvested, washed twice with ice-cold PBS, and fixed overnight at 4°C in pre-chilled 70% ethanol. Following two additional PBS washes, nuclear staining was performed by first treating samples with 100 μL RNase A (10 μg/mL) at 37°C for 30 min to remove RNA, followed by incubation with 400 μL propidium iodide (PI) solution under light-protected conditions at 37°C for 30 min.⁴⁵ Cell cycle distribution was then analyzed using a flow cytometer (BD Biosciences). The acquired data were processed through FlowJo software for the quantitative determination of cell population percentages in the G1, S, and G2/M phases.

mRNA decay assay

To evaluate mRNA degradation dynamics, transcriptional arrest was induced by 5 μg/mL actinomycin D (Gibco, Cat: 11805017) at specified time intervals (0, 2, 4 h, and 6 h) following siRNA-mediated gene silencing. Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Cat: 15596026) and subjected to reverse transcription using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Cat: 4387406). The remaining RNA level was analyzed by normalizing to baseline measurements at the 0 h timepoint.²⁶

RNA immunoprecipitation (RIP)

To investigate the potential interactions between the sclerostin/caprin1 and oncogenic mRNAs, we performed native RNA immunoprecipitation (RIP) using a Magna RIP Kit (Sigma-Aldrich, Cat: 17–700). Briefly, immunocomplexes were prepared by incubating anti-Flag antibody, anti-caprin1 antibody, or species-matched IgG control with magnetic Protein A/G beads in RIP buffer. Cell lysates were subsequently incubated with antibody-conjugated beads overnight at 4°C with gentle rotation. After washing six times, the bound RNAs were eluted and purified for reverse transcription, sequencing, and RT-qPCR analysis.²⁶

Protein complex modeling

The atomic model of the sclerostin-caprin1 complex was computationally generated using AlphaFold 3,⁴⁶ with canonical amino acid sequences serving as input parameters. The top-ranking structural prediction was selected based on optimal confidence metrics, specifically the predicted template modeling score (pTM) and interface template modeling score (ipTM) and subsequently subjected to comprehensive structural characterization. Molecular visualization and structural analysis were performed using UCSF ChimeraX software.⁴⁷ The protein-protein binding interface was defined by identifying all residues within a 5 Å contact distance threshold between the interacting partners.

In vivo distribution assay

The tissue distribution profile of Apc101/Scl-Ab was evaluated in MDA-MB-231 cells inoculated orthotopic mouse model. Mice received single subcutaneous doses of: (1) PBS vehicle control, (2) Cy3-labeled Apc101 (5 mg/kg), and (3) Cy3-labeled Scl-Ab (5 mg/kg), respectively. Following a 4-h circulation period, the mice were euthanized, followed by the collection of the major organs (heart, liver, spleen, lung, and kidney) along with tumor tissues for ex vivo fluorescence imaging.⁴⁸

Quantification and statistical analysis

The sample size, statistical methods, and relevant details are provided in the figure legends, main text, or methods section. Analyses were conducted in GraphPad Prism, with p < 0.05 considered significant. Data were expressed as mean ± standard deviation (SD). Error bars represent at least three independent experiments. Statistical comparisons were performed using unpaired t test or One-way ANOVA with Dunnett or Tukey post hoc test or two-way ANOVA with Dunnett or Bonferroni post hoc test. Sample sizes for animal studies were pre-determined based on power calculation, as detailed in our previous work.⁴⁹ ∗∗∗∗p < 0.0001; ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.05; ns, no significance.

Published: April 23, 2026