
==== Front
iScience
iScience
iScience
2589-0042
Elsevier

S2589-0042(24)01983-7
10.1016/j.isci.2024.110758
110758
Article
Identification of XD23 as a potent inhibitor of osteosarcoma via downregulation of DKK1 and activation of the WNT/β-catenin pathway
Xie Qian xqqx1996@163.com
126∗
Shen Yanni 3
Yang Yipei 3
Liang Jianhui 3
Wu Tailin 1
Hu Chun 4
Wang Yan yan.wang@siat.ac.cn
3∗∗
Tao Huiren huiren_tao@163.com
5∗∗∗
1 Department of Orthopedics, Shenzhen University General Hospital, Shenzhen 518055, China
2 Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging, National-Regional Key Technology Engineering Laboratory for Medical Ultrasound, School of Biomedical Engineering, Shenzhen University Medical School, Shenzhen 518060, China
3 Center for Translational Medicine Research and Development, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
4 Key Laboratory of Structure-based Drug Design & Discovery, Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, China
5 Department of Orthopaedics and Traumatology, The University of Hong Kong-Shenzhen Hospital, Shenzhen 518053, China
∗ Corresponding author xqqx1996@163.com
∗∗ Corresponding author yan.wang@siat.ac.cn
∗∗∗ Corresponding author huiren_tao@163.com
6 Lead contact

20 8 2024
20 9 2024
20 8 2024
27 9 11075819 2 2024
13 6 2024
14 8 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

Osteosarcoma, the most prevalent malignant bone tumor, is notorious for its aggressive growth and invasiveness. The highly mutable genome of osteosarcoma has made identifying a key oncogene challenging, hindering the development of targeted treatments. Our study validates the effectiveness of XD23, an anti-cancer agent we previously identified, in curbing osteosarcoma proliferation, metastasis, EMT differentiation, and bone destruction and promoting osteosarcoma apoptosis. It further elucidated that XD23 thwarts osteosarcoma by suppressing DKK1 expression, which in turn activates the WNT-β/Catenin pathway. This research presents the concrete evidence of DKK1’s involvement in osteosarcoma development, offering a foundation for the development of DKK1 inhibitors as novel treatments for this disease.

Graphical abstract

Highlights

• XD23 effectively inhibits osteosarcoma proliferation, metastasis, and bone destruction

• XD23 reduces DKK1 expression, activating the WNT-β/Catenin pathway

• The study confirms the role of DKK1 in osteosarcoma development

• Findings support DKK1 inhibitors as new therapies for osteosarcoma

Natural sciences; Biological sciences; Molecular biology; Cancer

Subject areas

Natural sciences
Biological sciences
Molecular biology
Cancer
Published: August 20, 2024
==== Body
pmcIntroduction

Osteosarcoma (OS) is a malignant bone tumor predominantly affecting children and adolescents.1 The established treatment protocol for OS includes neoadjuvant chemotherapy, surgical resection, and adjuvant chemotherapy.2 Regrettably, about 85% of OS cases are diagnosed at the metastatic stage, often in the lungs, resulting in a dismal five-year survival rate below 25%.3 First-line chemotherapy treatments for metastatic OS comprise high-dose methotrexate, cisplatin, doxorubicin, and ifosfamide, used either sequentially or in combination.4,5,6 Nonetheless, these medications carry substantial toxicity and often lead to resistance. Alternative chemotherapeutic agents like pamidronate have been tested but with disappointing outcomes.7 The genomic instability of OS, its varied subtypes, and the lack of clearly defined targets have obstructed the progress of targeted drug therapies.8,9 The stagnation in developing new treatments for metastatic OS over the last thirty years has stalled clinical advancements, underlining the critical need for more effective, targeted therapies.

Dickkopf-related protein 1 (DKK-1) is a glycoprotein mainly secreted by osteoblasts and bone cells.10,11 It belongs to the Dickkopf family and functions as an endogenous inhibitor of the canonical Wnt/β-catenin signaling pathway.12 DKK1 interrupts this pathway by competitively inhibiting the binding of Wnt to Frizzled-related proteins and by binding to LRP5/6 receptors and Kremen1/2 co-receptors, triggering their internalization.13 This inhibition is crucial in regulating tumor cell proliferation, differentiation, invasion, apoptosis, and metastasis.14,15,16 Promising clinical and preclinical results have been observed with several DKK1 inhibitors, such as DKN-01,17 BHQ-880,18 JS015,19 and IIIC3,20 in treating advanced solid tumors. High DKK1 expression levels in bone malignancies like multiple myeloma21 and metastatic bone tumors can disrupt bone formation and encourage resorption, leading to bone degradation.22 However, the role of DKK1 in OS remains uncertain.23 Some studies proposed that it functions as an oncogene,24,25 while others argued that it acts as an OS suppressor gene.26 There is still no definitive evidence of DKK1’s effects on OS cell proliferation, differentiation, migration, or its impact on bone integrity.

Given the significant involvement of DKK1 and the Wnt/β-catenin signaling pathway in bone-destructive diseases, it is reasonable to hypothesize that DKK1 may act as an oncogene in OS. However, more research is necessary to clarify DKK1’s precise function in OS and its viability as a treatment target. XD23, a small molecule compound designed and synthesized by our laboratory, and it has demonstrated broad-spectrum antitumor activity, as highlighted in our published research.27 In this study, we aim to evaluate the effectiveness of XD23 in inhibiting metastatic OS by targeting DKK1 and activating the WNT/β-catenin signaling pathway. Through extensive pharmacological experiments conducted at the genetic, cellular, and animal levels, we aim to provide compelling evidence supporting the use of DKK1 small molecule inhibitors as a promising treatment option for metastatic OS.

Results

XD23 suppresses cell proliferation and induces G0/G1 phase arrest in OS cells

In our lab, we synthesized a series of pyridine[2,3-day]pyrimidine compounds,26,27 with XD23 (Mol. Wt. = 471.18 g/mol, cLogP = 3.47, Figure 1A) emerging as the most effective against OS. We assessed the anti-proliferative effects of XD23 on three human OS cell lines—Saos2, MG63, and 143B—using CCK-8 assays, which showed IC50 values at 48h of 0.86 μM, 0.98 μM, and 1.48 μM, respectively. For comparison, normal human cell lines C28/I2 and HSF demonstrated CC50 values at 48h of 38.95 μM and 23.62 μM, respectively. This indicates a 16 to 45-fold selectivity for targeting cancer cells. At 0.5 μM, XD23 shows no cytotoxicity toward normal cells but inhibits the proliferation of three osteosarcoma cell lines by 5%–30%. Concentrations slightly above 0.5 μM exceed the IC50, while those slightly below have minimal effect. Hence, 0.5 μM XD23 was selected for further mechanistic studies (Figure 1B). Additionally, XD23’s ability to significantly reduce colony formation in OS cells further confirms its suppressive role in cell proliferation (Figures 1C–1E). Flow cytometry analysis showed a significant rise in the percentage of OS cells in the G0/G1 phase, suggesting that XD23 induces G0/G1 arrest (Figures 1F and 1G). Overall, the data robustly supports XD23’s capability to halt OS cell growth and trigger G0/G1 phase arrest.Figure 1 XD23’s in vitro effects on OS cell proliferation and G0/G1 phase arrest

(A) XD23’s chemical structure; (B) IC50 values for OS cell lines and CC50 for normal cells post XD23 treatment.

(C–E) Plate colony formation effects of XD23 and quantitative results in 143B (D) and MG63 cells (E).

(F and G) Flow cytometry analysis of the cell cycle in OS cells and quantitative outcomes. ∗p < 0.05; ∗∗p < 0.01. Data was analyzed by one sample t-test.

XD23 induces apoptosis in OS cells via mitochondrial and ER pathway

Next, the potential of XD23 to induce apoptosis in OS cells were probed. After 24 h of exposure to 0.5 μM XD23, mitochondrial permeability transition pores were compromised in two OS cell lines, indicated by a marked decrease in orange mitochondrial fluorescence (Figure 2A). Flow cytometry analysis revealed a dose-dependent increase in the number of early and late apoptotic cells in OS cells treated with XD23, with the percentages escalating from 3.52% to 10.8% and 1.21–29.1% respectively (Figures 2B and 2C). This data reinforces the pro-apoptotic effect of XD23.Figure 2 XD23 promotes apoptosis in OS cells by elevating ER Ca2+ level

(A) Mitochondrial membrane potential assay with JC-1 staining in OS cells post XD23 treatment for 24h or 48h. Scale bars = 10 μM.

(B and C) Annexin V-FITC/PI-stained OS cells analyzed by flow cytometry.

(D–F) Dose-dependent protein expression changes related to apoptosis in OS cells via western blotting. Normalized protein expression of WB in 143B cells (E) and MG63 cells (F).

(G) Fluro-4 AM detection of Ca2+ dynamics in OS cells treated with XD23 in 12h.

(H) ER staining in 143B cells treated with XD23. Scale bars = 10 μM.

(I) Western blotting of Calcinerin A and Calcinerin B protein expression after treated with XD23 for 24h or 48h.

(J) Normalized protein expression analysis of (I). ∗p < 0.05; ∗∗ <0.01; ∗∗∗p < 0.001. Data was analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test.

The apoptotic mechanism was further confirmed by increased caspase-3 activation, a key apoptosis regulator,28 in 143B and MG63 cells with ascending XD23 doses. PARP cleavage, which prompts caspase-3 to induce apoptosis,29 was observed in 143B cells but not in MG63 cells. XD23 also disrupted the Bcl-2 family protein balance by elevating the ratio of pro-apoptotic protein BAX to anti-apoptotic protein Bcl-2 in OS cells, thereby favoring apoptosis (Figures 2D–2F). These findings collectively suggest that XD23 initiates apoptosis in OS cells predominantly through the mitochondrial pathway.

Interestingly, we noted that XD23 stimulation activates caspase-12, an apoptosis-related protein uniquely present on the endoplasmic reticulum (ER),30 in both 143B and MG63 cells (Figures 2D–2F), implying that the ER pathway also contributes to apoptosis. The activation is further supported by the significant release of calcium ions from the ER calcium pump upon XD23 stimulation. As depicted in Figures 2G and S1, this stimulation escalates the intracellular calcium ion concentration in 143B and MG63 cells.

To confirm that XD23 induces apoptosis in OS cells via the ER stress pathway, we first noted structural changes in the ER using an ER fluorescent probe (Figures 2H and S2). Additionally, we observed a significant upregulation in both the A and B subunits of Calcineurin (Figures 2I and 2J), a protein that responds to calcium ion concentration and indicates ERS activation.31,32 These findings suggest that XD23 can induce ER stress and promote apoptosis in OS cells by elevating calcium ion concentration.

XD23 attenuates OS cells migration by suppressing EMT differentiation

Extensive research has highlighted a direct link between the overexpression of EMT-related transcription factors in OS and the enhanced migratory and invasive capabilities of OS cells, ultimately driving metastasis. Our transwell migration assay results demonstrate that 0.1 μM XD23 significantly reduces OS cell migration (Figure 3A). Furthermore, a wound healing assay revealed that treating 143B cells with 0.5 μM XD23 for 6 h led to a notable 34% reduction in wound closure, indicating a potent suppression of OS cell invasion by XD23 (Figures 3B and 3C).Figure 3 XD23 deters OS cells migration by impeding EMT differentiation

(A) Transwell migration assay showing concentration-dependent inhibition of 143B cell invasion by XD23.

(B) Wound healing assay demonstrating the inhibitory effect of XD23 (0.5 μM) on 143B cell migration and (C and C) the normalized wounding areas. Scale bars = 200 μM.

(D and E) Changes of mRNA expression related to EMT differentiation after XD23 (0.5 μM) stimulation in 143B cells and MG63 cells.

(F) E-cadherin and N-cadherin protein expression after treated with XD23 (0.5 μM) for 24h and 48h.

(G) Normalized expression of E-cadherin and N-cadherin protein expression in western blotting.

(H) Immunofluorescence staining for E-cadherin and N-cadherin in 143B and MG63 cells post-XD23 treatment. Scale bars = 10 μM ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Data was analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test.

Further investigation into the mechanism revealed that XD23 affects the transcriptional control of EMT-related genes in OS cells (Figures 3D and 3E). Our findings showed that post 48 h of stimulation with 0.5 μM XD23, there was a remarkable 8- to 16-fold increase in the expression of the EMT marker gene E-cadherin, while N-cadherin expression significantly dipped by 3- to 5-fold. Nevertheless, the expression of Vimentin, TJP-1, or Snail1 genes remained unaffected. These observations were further corroborated by Western blot (Figures 3F and 3G) and immunofluorescence experiments (Figure 3H), confirming that XD23 indeed upregulates E-cadherin expression and downregulates N-cadherin protein levels. In summary, our experimental data compellingly suggest that XD23 impedes the invasive and migratory tendencies of OS cells by regulating their EMT differentiation.

RNA-seq discloses XD23 inhibits OS by inhibiting DKK1 to activate Wnt/β-catenin signaling pathway

To unravel the mechanism behind XD23’s impact on OS cell functions, including proliferation, apoptosis, migration, and differentiation, we performed RNA sequencing on total RNA from 143B cells treated with 0.5 μM XD23 for 48 h. The resulting volcano plot highlighted significant transcriptomic shifts, with 243 genes upregulated and 329 genes downregulated, notably including a pronounced decrease in DKK1 (Figure 4A). Gene Ontology (GO) analysis further implicated XD23 in modulating various tumor-associated pathways, with a particular emphasis on alterations in calcium and Wnt signaling pathways (Figure 4B). Heatmap analysis of gene expression linked to the DKK1-Wnt axis confirmed an induction of Wnt/β-catenin signaling-related genes following DKK1 suppression (Figures 4C and 4D). Additionally, genes interacting with DKK1, such as Wnt5A/B and FZD5/6, and those involved in the Wnt-Ca2+ pathway, showed increased expression, a finding substantiated by qPCR results (Figure 4E).Figure 4 XD23 inhibits OS by downregulating DKK1 to activate Wnt signaling pathway

(A) The Volcano plot showing gene expression differences between XD23-treated and control groups (n = 3 biologically independent cell samples). Significant genes identified using Cuffdiff, with upregulated transcripts marked in red and downregulated in blue.

(B) GO analysis of biological processes affected by regulated genes.

(C–G) Heatmap of Wnt pathway gene expression changes. Representative gene FPKM values (D) and qPCR validation (E). Western blotting results (F) and the normalized quantitative results (G) of WNT signaling pathway related proteins in 143B cells after XD23 stimulation.

(H–J) The immunofluorescence analysis of β-catenin in 143B cells after treated with XD23. Scale bars = 10 μM. Western blotting results (I) and the normalized quantitative results (J) of overexpression Dkk1 transfected 143B cells. ####p < 0.0001 vs. Dkk1+. ∗∗∗∗p < 0.0001 vs. WT.

(K) Comparison of cell cytotoxicity results of XD23 on WT-type 143B cells and Dkk1 overexpressing 143B cells. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. ns = no significance. Data was analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test.

Western blot analysis showed that XD23 reduces DKK1 protein levels and increases β-catenin and its phosphorylated form at 0.2 μM and 0.5 μM concentrations (Figures 4F–4H), indicating that XD23 activates the Wnt/β-catenin pathway by inhibiting DKK1 in vitro. To explore the role of DKK1 in XD23’s anti-OS activity, Dkk1 transfected overexpressing 143B cells were generated (Figure 4I). These cells showed high DKK1 protein levels with reduced β-catenin, which normalized after XD23 treatment, resembling wild-type 143B cells (Figures 4I and 4J). Comparing the growth-inhibitory effects of XD23 on Dkk1-overexpressing cells to wild-type revealed a 2.9-fold decrease in efficacy for the former, underscoring DKK1’s significance in XD23’s mechanism (Figure 4K). In conclusion, our findings strongly indicate that XD23’s anti-OS activity in vitro is due to a pharmacological mechanism that involves Wnt/β-catenin pathway activation through DKK1 inhibition.

XD23 suppresses orthotopic OS tumor growth and metastasis in vivo

In our investigation of XD23’s efficacy against metastatic OS, we developed an orthotopic mouse model by inoculating rat-derived OS cells (UMR-106) into the distal tibia bone marrow. Commencing one-week post-inoculation, XD23 was administered orally at 15 mg/kg and 10 mg/kg for high and low doses, respectively, 2–3 times weekly over a four-week period As a control, we used high-dose methotrexate (MTX) at 10 mg/kg, with a 5 mg/kg folic acid supplement 24 h after MTX administration (Figure 5A).Figure 5 XD23’s impact on anti-metastatic OS in vivo

(A) Orthotopic metastatic OS mouse model experimental design.

(B) OS mouse survival rates per group.

(C) Mouse body weight fluctuations during the experiment.

(D) Images of OS in the right limb of each mouse group.

(E) Lung H&E staining representations for each group.

(F) Micro-CT scans of each group’s right tibia.

The Model group, receiving saline, began to show mortality from day 15, with no survivors by the end of the treatment period. The MTX group exhibited mortality beginning on day 19, with a survival rate of 25% at the end of the study. By contrast, the XD23-treated mice demonstrated improved survival, with no deaths until days 23 and 27 for low and high doses, respectively, culminating in a 75% survival rate, a marked enhancement over MTX (Figure 5B). Additionally, body weight changes showed decreased weight in all OS groups compared to the Sham group. Notably, the MTX group experienced a sharp drop in the final week. In contrast, the XD23 groups maintained their body weight more effectively (Figure 5C). Histological analysis with hematoxylin and eosin (H&E) staining of visceral organs showed no discernible organ toxicity from XD23, confirming its safety in vivo (Figure S3). The XD23 groups, especially at the higher dose, showed a notable reduction in hindlimb sarcoma volume versus the MTX group, underscoring XD23’s potent anti-proliferative effects (Figure 5D). Examinations of the lungs postmortem revealed extensive tumor formation in the control group, signifying advanced pulmonary metastasis. However, gross (Figure S4) and H&E (Figure 5E) inspections of the XD23 groups indicated significantly less metastatic spread compared to the MTX group, highlighting XD23’s potential in curbing metastatic progression in OS. Therefore, XD23 has proven to suppress OS proliferation and metastasis in vivo, showing promise in limiting the advancement and dissemination of lung metastatic OS. Further studies are needed to confirm if its effects are due to DKK1 inhibition in vivo.

XD23 mitigates OS-induced skeletal damage in vivo

Micro-CT scans of the right tibia in each mouse group (Figure 5F) revealed severe cortical bone erosion in the OS-affected mice, characterized by thinning bone shafts and visible cavities, bordering on fracture. In contrast, mice treated with MTX demonstrated an appreciable increase in bone mass compared to the model group. Most strikingly, XD23 treatment yielded pronounced cortical bone thickening and structural reinforcement, suggesting a role for the compound in halting OS growth and reducing bone loss. Furthermore, in vitro osteoclast culture experiments revealed that high concentrations of XD23 (10μM and 3μM) can significantly inhibit osteoclast differentiation (Figure S5). To encapsulate, these results support XD23’s potential as a therapeutic agent in alleviating OS-induced skeletal deterioration.

XD23 curbs orthotopic OS tumor through DKK1 inhibition and WNT/β-catenin signaling pathway activation

Histological examination of H&E-stained tibial tumor sections from each mouse group revealed densely packed cells within a thin fibrous capsule, indicative of OS (Figure 6A). Further staining experiments will be conducted on in situ OS and pulmonary metastatic OS samples to confirm if XD23, as hypothesized, also downregulates DKK1 to activate the Wnt/β-catenin signaling pathway in vivo, thereby suppressing OS cell proliferation and metastasis, promoting apoptosis, and mitigating bone destruction.Figure 6 XD23’s role in WNT/β-Catenin activation in vivo

(A) TUNEL staining of OS tissue slices from each mouse groups. Scale bars = 1 mm.

(B) TUNEL staining fluorescence intensity average.

(C) H&E staining images of OS tissue from each mouse group Scale bars = 1 mm.

(D–F) DKK1 and β-catenin IHC staining in OS tissue. Scale bars 50 μm. The relative positive area ratio of IHC staining for DKK1 (E) and β-catenin (F) in OS slices.

(G–I) IHC staining of DKK1 and β-catenin in lung slices of mice in each group. Scale bars = 50 μm. The relative positive area ratio of IHC staining for DKK1 (H) and β-catenin (I) in lung slices.

(J) XD23’s mechanism of action against OS in vivo. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001. ns = no significance. Data was analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test.

Initial TUNEL assays demonstrated that high-dose XD23 markedly induced apoptosis in OS cells in tumor OS cells, with effectiveness similar to lower doses of MTX (Figures 6B and 6C). Immunohistochemistry of tibial and pulmonary OS samples showed marked DKK1 suppression in the XD23-treated group compared to the Model groups (Figures 6D and 6E), while β-catenin levels were sustained in the Model and high-dose XD23 groups (Figures 6D and 6F). Lung metastases displayed high DKK1 levels, which XD23 significantly reduced compared to MTX. Conversely, β-catenin expression was minimal in the MTX group but increased with high-dose XD23 (Figures 6G–6I). These findings imply that XD23 modulates the Wnt/β-catenin pathway by reducing DKK1 expression, enhancing β-catenin activity, and contributing to bone preservation (Figure 6J).

Discussion

DKK1 is crucial in the Wnt/β-catenin signaling pathway, significantly affecting the onset, progression, and therapeutic response in OS.33,34 High DKK1 expression correlates with worsened malignancy and prognosis in OS,23,35 but its over-suppression could inadvertently trigger oncogenic Wnt/β-catenin signaling.36 Thus, DKK1-targeted therapies must be finely tuned to effectively check tumor growth and dissemination.

This research presents the anticancer agent XD23, which modulates DKK1, thereby influencing Wnt/β-catenin signaling. XD23’s antitumor potency correlates with DKK1 expression levels. It curbs proliferation in various OS cell lines, induces cell-cycle arrest, and demonstrates selective toxicity toward OS cells compared to non-cancerous cells. Remarkably, XD23 significantly hampers proliferation in DKK1-overexpressing 143B cells, hinting that its action might also involve additional oncogenic pathways.

Contrary to the norm where aberrant Wnt pathway activation fosters tumor growth and metastasis by blocking apoptosis,37 this study shows that DKK1 inhibition by XD23 triggers apoptosis in OS cells in a dose-dependent manner. It elevates ER calcium and disrupts mitochondrial membrane potential, suggesting involvement of both mitochondrial and ER pathways. This is the inaugural demonstration of apoptosis promotion through DKK1 inhibition in OS, bolstering the potential of DKK1 antagonists in OS treatment.

Considering the high mortality of OS due to lung metastases,34 it is vital to understand how Wnt pathway modulation affects OS cell behavior. XD23 effectively hinders OS cell migration, invasion, and epithelial-mesenchymal transition in vitro and significantly reduces lung metastasis in OS animal models, indicating that DKK1 inhibition and consequent Wnt/β-catenin pathway activation may impede OS cell metastasis.

The Wnt/β-catenin pathway is indispensable for differentiating bone marrow mesenchymal stem cells into osteoblasts,38 and its disruption can cause bone diseases like osteoporosis.39 In advanced OS, bone destruction is a significant hurdle. Our findings indicate that XD23 activates the Wnt/β-catenin pathway via DKK1 inhibition in an in situ OS model, unlike methotrexate, potentially conserving bone mass and offering a therapeutic edge in preventing tumor-induced bone loss.

In summary, our investigation validates the antineoplastic properties of XD23, a compound that targets OS by downregulating DKK1 and activating the Wnt/β-catenin pathway. XD23 selectively halts OS cell proliferation, especially in the DKK1-overexpressing 143B cell line. Its antitumor effect is apoptosis-driven, engaging mitochondrial and ER pathways. Moreover, XD23 markedly impedes OS cell migration, invasion, and EMT, contributing to reduced lung metastasis in vivo. Divergent from agents like methotrexate, XD23 maintains bone integrity by modulating the Wnt/β-catenin pathway, presenting a benefit in averting tumor-associated bone deterioration. These findings support the clinical investigation of DKK1 inhibitors as a strategy for OS treatment.

Limitations of the study

While this study has systematically investigated the inhibitory mechanism of XD23 on OS, we must acknowledge certain limitations. First, further in-depth research is required to fully understand how XD23 inhibits osteoclast-induced bone resorption. This study has only preliminarily explored its effect on inhibiting osteoclast differentiation, and a more detailed investigation of the mechanisms involved is necessary. Second, although this study has identified the downstream proteins of XD23, there is a need for further exploration of its upstream target proteins. Despite these limitations, the conclusions drawn in this study remain valid. We plan to address these issues in future research to provide more comprehensive evidence supporting the use of XD23 in OS treatment.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Q.X. (xqqx1996@163.com).

Materials availability

The study did not generate new unique reagents.

Data and code availability

• Raw data and processed data were uploaded to Mendeley Data and are available via https://doi.org/10.17632/kb6g5n2nnz.1. RNA-seq raw data were uploaded in the GEO datasets and are available via GSE262143.

• This study did not generate original code.

• Any additional information required to reanalyzes the data reported in this paper is available form the lead contact upon request.

Acknowledgments

Funding: This work was supported by 10.13039/501100001809 National Natural Science Foundation of China (82174033 to Y.W., 81970761 to H.R.T.).

Author contributions

Conceptualization, Q.X., H.R.T., and Y.W.; Methodology, Q.X. and Y.N.S.; Investigation, Y.P.Y. and J.H.L.; Validation and Data Curation, Y.N.S. and J.H.L.; Resources, C.H.; Writing – Original Draft, Q.X. and Y.W.; Writing – Review and Editing, Q.X.; Supervision, H.R.T.; Funding Acquisition, H.R.T. and Y.W.

Declaration of interests

The authors declare that they have no known competing financial interests of personal relationships that could have appeared to influence the work reported in this paper.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Antibodies	
	
Rabbit monoclonal to E Cadherin	Abcam	Cat#ab18203	
Mouse monoclonal to E Cadherin	Abcam	Cat#ab231303	
Rabbit monoclonal to N cadherin	Abcam	Cat#ab18203	
Rabbit monoclonal to DKK1	Abcam	Cat#ab307367	
Rabbit monoclonal to β-catenin	Abcam	Cat#ab32572	
Rabbit monoclonal to β-catenin phosphor(active) S45	Abcam	Cat#ab30561	
Goat Anti-Rabbit IgG H&L (HRP)	Abcam	Cat#ab6721	
Goat Anti-Mouse IgG H&L (HRP)	Abcam	Cat#ab205719	
Goat Anti-Rabbit Alexa Fluor® 488	Abcam	Cat#ab150077	
Goat Anti-Mouse Alexa Fluor® 594	Abcam	Cat#ab150116	
Rabbit monoclonal to GAPDH	Abcam	Cat#ab181602	
Rabbit monoclonal to PARP	Cell Signaling Technology	Cat#9542	
Rabbit polyclonal to cleaved-PARP	Cell Signaling Technology	Cat#9548	
Rabbit polyclonal to caspase-12	Cell Signaling Technology	Cat#35965	
Rabbit polyclonal to BAX	Cell Signaling Technology	Cat#2772	
Rabbit polyclonal to Bcl-2	Cell Signaling Technology	Cat#3498	
Rabbit polyclonal to caspase-3	Cell Signaling Technology	Cat#9662	
Rabbit polyclonal to cleaved-caspase-3	Cell Signaling Technology	Cat#9661	
	
Bacterial and virus strains	
	
Human-DKK1 plasmid	Hanyin company	N/A	
	
Biological samples	
	
MICE OS tumor	This paper	N/A	
MICE lung/spleen/heart/kidney/liver	This paper	N/A	
	
Chemicals, peptides, and recombinant proteins	
	
XD23	Laboratory Synthesis from Shenyang Pharmaceutical University	https://www.sciencedirect.com/science/article/pii/S0223523422004810	
MTX	Sigma	PHR1396	
	
Deposited data	
	
Raw RNAseq data	GEO datasets	GSE262143	
Original, unprocessed data	Mendeley data	https://doi.org/10.17632/kb6g5n2nnz.1	
	
Experimental models: Cell lines	
	
Human osteosarcoma cell lines 143B	Stem Cell Bank of the Chinese Academy of Sciences	N/A	
Human osteosarcoma cell lines MG63	Stem Cell Bank of the Chinese Academy of Sciences	N/A	
Human skin fibroblasts HSF	Stem Cell Bank of the Chinese Academy of Sciences	N/A	
human chondrocytes C28/I2	Stem Cell Bank of the Chinese Academy of Sciences	N/A	
Rat osteosarcoma cell line UMR-106	Stem Cell Bank of the Chinese Academy of Sciences	N/A	
	
Oligonucleotides	
	
See Table S1 for primer sequences	N/A	N/A	
	
Software and algorithms	
	
ImageJ	NIH	Version 1.52	
GraphPad Prism	GraphPad	Version 9.0.0	

Experimental model and study participant details

Cell lines

Human osteosarcoma cell lines 143B, MG63 and Sasos2, rat osteosarcoma cell line UMR-106, human skin fibroblasts (HSF), and normal human chondrocytes (C28/I2) were procured from the Stem Cell Bank of the Chinese Academy of Sciences in Shenzhen, China. The 143B, MG63 and Sasos2 cell lines were propagated in RPMI-1640 medium (Corning, US), while HSF, C28/I2, and UMR-106 cell lines were grown in DMEM-HG medium (Corning, US). Both media were enriched with 10% fetal bovine serum (Gibco, US) and 1% penicillin-streptomycin (Gibco, US). Cultures were maintained at 37°C in a 5% CO2 atmosphere.

OS tumor mice model

In the orthotopic OS tumor model, male BALB/c nude mice (4–5 weeks old) were kept in a pathogen-free environment at SIAT and divided into 5 groups of 6: Vehicle, Sham, MTX, XD23-Low (10 mg/kg), and XD23-High (15 mg/kg). The Sham group received saline orally, while the MTX group served as a positive control with methotrexate.

UMR-106 cells were cultured, trypsinized with 0.25% trypsin, and suspended in PBS at 2 × 10ˆ6 cells/mL. After anesthetizing with Zoletil-50 (Virbac, FR), a mouse’s right hindlimb was disinfected and a syringe inserted into the tibia. Stable placement confirmed accurate needle positioning. A 30μL cell suspension was injected into the tibia of the mice, except for the Vehicle group, which received saline. Mice were monitored for body weight and tumor formation, treated after 2 weeks for 28 days, euthanized after cardiac perfusion and then dissected for organ fixation in 10% neutral formalin.

All animal handling and use was approved by Institutional Animal Care and Use Committee (IACUC) at SIAT on May 1, 2022. The registration number is SIAT-IACUC-220412-YGS-WY-A2136.

Method details

CCK-8 assay

Cell viability of XD23 on 143B, MG63, HSF, and C28/I2 cells was evaluated using the CCK-8 assay (DOJINDO, JP). We seeded about 5 × 10ˆ3 cells per well in a 96-well plate. After cell adhesion, we introduced 200 μL of fresh medium with various XD23 concentrations. Post a 48h incubation, we added 10 μL of CCK-8 solution to each well and incubated for another hour. The plate was then gently shaken for around 20s, and absorbance at 450 nm was measured using a microplate reader.

Plate clone formation assay

About 8 × 10ˆ2 143B and MG63 cells were cultured in a 6-well plate, with the medium enhanced by XD23 in concentrations ranging from 0.02 μM to 1.0 μM for 7 days post-adhesion. Afterward, the wells were PBS-rinsed and set with 4% paraformaldehyde for 15 min, followed by staining with 1% crystal violet dye solution (Solarbio, G1062#, China).

Cell-cycle arrest analysis

143B cells at 70–80% confluence in 12-well plates were treated with 0.1 μM–1.0 μM XD23 at 37°C for 48 h. Post pancreatin digestion and PBS wash, cells were fixed in 50% ethanol and stained with propidium iodide solution (Sigma, US). Flow cytometry (Beckman, 524C CytoFLEX, US) and ModFit software analyzed DNA content.

Wounding healing assay

1 × 10ˆ6 143B cells were plated in 6-well plates with ibidi Culture-Inserts (ibidi, DE) and cultivated in serum-free medium with 0.5 μM XD23 for 24 h. Sequential images of the scratch were taken every 6 h.

Transwell migration assay

For the Transwell migration assay, a 6.5 mm Transwell with an 8.0 μm PET insert (Corning, 3464#, US) was placed in a 24-well plate and coated with a Matrigel Matrix mixture. Culture medium containing various XD23 doses was added, and 143B cells were seeded in the insert’s upper chamber. After 48 h of incubation to allow migration, non-migrated cells were removed, and the rest were fixed, stained, and counted.

Real-time quantitative PCR

1 × 10ˆ5 143B and MG63 cells were seeded in a 12-well plate at 70–80% confluence, followed by 0.5 μM XD23 treatment for 24 or 48 h. Cells were lysed with TRIZOL (Thermo Fisher, US), reverse transcribed using PrimeScript RT Master Mix (TaKaRa, RR036A#, JP), and RT-qPCR was performed on a LightCycler96 with TB Green dye (TaKaRa, RR820A#, JP), using primers listed in Table S1.

Western Blotting

Protein lysates from 143B and MG63 cells treated with XD23 for 48 h were extracted using RIPA buffer (Thermo Fisher, US), separated by 10% SDS-PAGE, and transferred to a PVDF membrane (Millipore, GER). The membrane was blocked with 2.5% skimmed milk in TBST and incubated overnight at 4°C with primary antibodies including PARP, cleaved PARP, Caspase-12, BAX, Bcl-2, Caspase-3, cleaved caspase-3, GAPDH, N-cadherin, E-cadherin, DKK1, and WNT5A.

Micro-CT

All mice limbs were positioned in a 34 mm scanning tube and imaged with a μ-CT device (Scanco Microct, Switzerland) at 18 μm resolution, using 55 kV/200μA settings40

Histological analysis

The internal organs were embedded in paraffin and sliced into 6-μm-thick sections. After dewaxed with xylene, rehydrated with ethanol, the sections were stained with Hematoxylin and Eosin (H&E, Beyotime, CN) as described previously.41 Protein expression levels of DKK1 and β-catenin were evaluated by probing with corresponding antibodies on the sections.

RNA-seq and data analysis

143B cells were seeded in the 6-well plates and incubated for 48h after stimulated with 0.5 μM XD23. Then cells were lysed by TRIZOL reagent and sent to BGI Genomics institution for RNA extraction and bulk mRNA sequencing. The Raw sequencing data analysis were performed as described previously40

Plasmid transfection

Human-DKK1 plasmid was obtained from Hanyin company (Wuhan, CN). Lipofectamine 3000 (Invitrogen, US) was used as transfection reagent. 5 × 10ˆ5 143B cells were seeded on 6-well plate and then performed the transfection under the manuals after 70% cells confluence. Transfection efficiency was verified by qPCR and fluorescence microscopy.

Quantification and statistical analysis

All experiments were conducted in triplicate, with data presented as mean ± SD from representative experiments (n ≥ 3). Significance was denoted as ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, or ∗p < 0.05, analyzed using GraphPad Prism software (CA, US).

Supplemental information

Document S1. Figures S1‒S9 and Table S1

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2024.110758.
==== Refs
References

1 Isakoff M.S. Bielack S.S. Meltzer P. Gorlick R. Osteosarcoma: Current Treatment and a Collaborative Pathway to Success J. Clin. Oncol. 33 2015 3029 3035 10.1200/jco.2014.59.4895 26304877
2 Harrison D.J. Geller D.S. Gill J.D. Lewis V.O. Gorlick R. Current and future therapeutic approaches for osteosarcoma Expert Rev. Anticancer Ther. 18 2018 39 50 10.1080/14737140.2018.1413939 29210294
3 Bacci G. Rocca M. Salone M. Balladelli A. Ferrari S. Palmerini E. Forni C. Briccoli A. High Grade Osteosarcoma of the Extremities With Lung Metastases at Presentation: Treatment With Neoadjuvant Chemotherapy and Simultaneous Resection of Primary and Metastatic Lesions J. Surg. Oncol. 98 2008 415 420 10.1002/jso.21140 18792969
4 Meyers P.A. Schwartz C.L. Krailo M. Kleinerman E.S. Betcher D. Bernstein M.L. Conrad E. Ferguson W. Gebhardt M. Goorin A.M. Osteosarcoma: A randomized, prospective trial of the addition of ifosfamide and/or muramyl tripeptide to cisplatin, doxorubicin, and high-dose methotrexate J. Clin. Oncol. 23 2005 2004 2011 10.1200/jco.2005.06.031 15774791
5 O'Day K. Gorlick R. Novel therapeutic agents for osteosarcoma Expert Rev. Anticancer Ther. 9 2009 511 523 10.1586/era.09.7 19374604
6 Ando K. Mori K. Corradini N. Redini F. Heymann D. Mifamurtide for the treatment of nonmetastatic osteosarcoma Expert Opin. Pharmacother. 12 2011 285 292 10.1517/14656566.2011.543129 21226638
7 Meyers P.A. Healey J.H. Chou A.J. Wexler L.H. Merola P.R. Morris C.D. Laquaglia M.P. Kellick M.G. Abramson S.J. Gorlick R. Addition of Pamidronate to Chemotherapy for the Treatment of Osteosarcoma Cancer 117 2011 1736 1744 10.1002/cncr.25744 21472721
8 Smida J. Baumhoer D. Rosemann M. Walch A. Bielack S. Poremba C. Remberger K. Korsching E. Scheurlen W. Dierkes C. Genomic Alterations and Allelic Imbalances Are Strong Prognostic Predictors in Osteosarcoma Clin. Cancer Res. 16 2010 4256 4267 10.1158/1078-0432.Ccr-10-0284 20610556
9 Grignani G. Palmerini E. Ferraresi V. D'Ambrosio L. Bertulli R. Asaftei S.D. Tamburini A. Pignochino Y. Sangiolo D. Marchesi E. Sorafenib and everolimus for patients with unresectable high-grade osteosarcoma progressing after standard treatment: a non-randomised phase 2 clinical trial Lancet Oncol. 16 2015 98 107 10.1016/s1470-2045(14)71136-2 25498219
10 Chu H.Y. Chen Z. Wang L. Zhang Z.K. Tan X. Liu S. Zhang B.T. Lu A. Yu Y. Zhang G. Dickkopf-1: A Promising Target for Cancer Immunotherapy Front. Immunol. 12 2021 658097 10.3389/fimmu.2021.658097 34093545
11 Diarra D. Stolina M. Polzer K. Zwerina J. Ominsky M.S. Dwyer D. Korb A. Smolen J. Hoffmann M. Scheinecker C. Dickkopf-1 is a master regulator of joint remodeling Nat. Med. 13 2007 156 163 10.1038/nm1538 17237793
12 Niida A. Hiroko T. Kasai M. Furukawa Y. Nakamura Y. Suzuki Y. Sugano S. Akiyama T. DKK1, a negative regulator of Wnt signaling, is a target of the β-catenin/TCF pathway Oncogene 23 2004 8520 8526 10.1038/sj.onc.1207892 15378020
13 Bafico A. Liu G. Yaniv A. Gazit A. Aaronson S.A. Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/Arrow Nat. Cell Biol. 3 2001 683 686 10.1038/35083081 11433302
14 Igbinigie E. Guo F. Jiang S.W. Kelley C. Li J. Dkk1 involvement and its potential as a biomarker in pancreatic ductal adenocarcinoma Clin. Chim. Acta 488 2019 226 234 10.1016/j.cca.2018.11.023 30452897
15 Zhang Y. Liang K. Zhou X. Zhang X. Xu H. Dai H. Song X. Yang X. Liu B. Shi T. Wei J. Combination therapy of DKK1 inhibition and NKG2D chimeric antigen receptor T cells for the treatment of gastric cancer Cancer Sci. 114 2023 2798 2809 10.1111/cas.15828 37151176
16 Zhu G. Song J. Chen W. Yuan D. Wang W. Chen X. Liu H. Su H. Zhu J. Expression and Role of Dickkopf-1 (Dkk1) in Tumors: From the Cells to the Patients Cancer Manag. Res. 13 2021 659 675 10.2147/cmar.S275172 33536782
17 Goyal L. Sirard C. Schrag M. Kagey M.H. Eads J.R. Stein S. El-Khoueiry A.B. Manji G.A. Abrams T.A. Khorana A.A. Phase I and Biomarker Study of the Wnt Pathway Modulator DKN-01 in Combination with Gemcitabine/Cisplatin in Advanced Biliary Tract Cancer Clin. Cancer Res. 26 2020 6158 6167 10.1158/1078-0432.Ccr-20-1310 32878766
18 Fulciniti M. Tassone P. Hideshima T. Vallet S. Nanjappa P. Ettenberg S.A. Shen Z. Patel N. Tai Y.T. Chauhan D. Anti-DKK1 mAb (BHQ880) as a potential therapeutic agent for multiple myeloma Blood 114 2009 371 379 10.1182/blood-2008-11-191577 19417213
19 Noh J.G. Jeon H.E. So J.S. Chang W.S. Effects of the Bradyrhizobium japonicum waaL(rfaL) Gene on Hydrophobicity, Motility, Stress Tolerance, and Symbiotic Relationship with Soybeans Int. J. Mol. Sci. 16 2015 16778 16791 10.3390/ijms160816778 26213919
20 González S. Oh D. Baclagon E.R. Zheng J.J. Deng S.X. Wnt Signaling Is Required for the Maintenance of Human Limbal Stem/Progenitor Cells In Vitro Invest. Ophthalmol. Vis. Sci. 60 2019 107 112 10.1167/iovs.18-25740 30640975
21 Dun X. Jiang H. Zou J. Shi J. Zhou L. Zhu R. Hou J. Differential expression of DKK-1 binding receptors on stromal cells and myeloma cells results in their distinct response to secreted DKK-1 in myeloma Mol. Cancer 9 2010 247 10.1186/1476-4598-9-247 20846389
22 Ke H.Z. Richards W.G. Li X. Ominsky M.S. Sclerostin and Dickkopf-1 as Therapeutic Targets in Bone Diseases Endocr. Rev. 33 2012 747 783 10.1210/er.2011-1060 22723594
23 Lee N. Smolarz A.J. Olson S. David O. Reiser J. Kutner R. Daw N.C. Prockop D.J. Horwitz E.M. Gregory C.A. A potential role for Dkk-1 in the pathogenesis of osteosarcoma predicts novel diagnostic and treatment strategies Br. J. Cancer 97 2007 1552 1559 10.1038/sj.bjc.6604069 17987039
24 Özgür A. Investigation of anticancer activities of STA-9090 (ganetespib) as a second generation HSP90 inhibitor in Saos-2 osteosarcoma cells J. Chemother. 33 2021 554 563 10.1080/1120009x.2021.1908650 33794753
25 Goldstein S.D. Trucco M. Bautista Guzman W. Hayashi M. Loeb D.M. A monoclonal antibody against the Wnt signaling inhibitor dickkopf-1 inhibits osteosarcoma metastasis in a preclinical model Oncotarget 7 2016 21114 21123 27049730
26 Shen Y. Xie Q. Wang Y. Liang J. Jiang C. Liu X. Wang Y. Hu C. Design, synthesis and anti-osteosarcoma activity study of novel pyrido [2, 3-d] pyrimidine derivatives by inhibiting DKK1-Wnt/β-catenin pathway Bioorg. Chem. 141 2023 106848 37716273
27 Xie Q. Shen Y. Meng Y. Liang J. Xu J. Liang S. Liu X. Wang Y. Hu C. Pyrido 2,3-d pyrimidine-2,4(1H,3H)-dione derivatives as RAF-MEK-ERK pathway signaling pathway blockers: Synthesis, cytotoxic activity, mechanistic investigation and structure-activity relationships Eur. J. Med. Chem. 240 2022 114579 10.1016/j.ejmech.2022.114579 35797896
28 Porter A.G. Jänicke R.U. Emerging roles of caspase-3 in apoptosis Cell Death Differ. 6 1999 99 104 10200555
29 Boulares A.H. Yakovlev A.G. Ivanova V. Stoica B.A. Wang G. Iyer S. Smulson M. Role of poly (ADP-ribose) polymerase (PARP) cleavage in apoptosis: caspase 3-resistant PARP mutant increases rates of apoptosis in transfected cells J. Biol. Chem. 274 1999 22932 22940 10438458
30 Hitomi J. Katayama T. Taniguchi M. Honda A. Imaizumi K. Tohyama M. Apoptosis induced by endoplasmic reticulum stress depends on activation of caspase-3 via caspase-12 Neurosci. Lett. 357 2004 127 130 15036591
31 Bonilla M. Nastase K.K. Cunningham K.W. Essential role of calcineurin in response to endoplasmic reticulum stress Embo J. 21 2002 2343 2353 10.1093/emboj/21.10.2343 12006487
32 Bousette N. Chugh S. Fong V. Isserlin R. Kim K.H. Volchuk A. Backx P.H. Liu P. Kislinger T. MacLennan D.H. Constitutively active calcineurin induces cardiac endoplasmic reticulum stress and protects against apoptosis that is mediated by α-crystallin-B Proc. Natl. Acad. Sci. USA 107 2010 18481 18486 10.1073/pnas.1013555107 20937869
33 Zhao X. Sun S. Xu J. Luo Y. Xin Y. Wang Y. MicroRNA-152 inhibits cell proliferation of osteosarcoma by directly targeting Wnt/β-catenin signaling pathway in a DKK1-dependent manner Oncol. Rep. 40 2018 767 774 29845282
34 Lin C.H. Ji T. Chen C.-F. Hoang B.H. Wnt signaling in osteosarcoma Adv. Exp. Med. Biol. 804 2014 33 45 24924167
35 Du X. Yang J. Yang D. Tian W. Zhu Z. The genetic basis for inactivation of Wnt pathway in human osteosarcoma BMC Cancer 14 2014 450 24942472
36 Cai Y. Cai T. Chen Y. Wnt pathway in osteosarcoma, from oncogenic to therapeutic J. Cell. Biochem. 115 2014 625 631 24190862
37 Cai Y. Mohseny A.B. Karperien M. Hogendoorn P.C.W. Zhou G. Cleton-Jansen A.M. Inactive Wnt/β-catenin pathway in conventional high-grade osteosarcoma J. Pathol. 220 2010 24 33 19882675
38 Yu F.-X. Hu W.-J. He B. Zheng Y.-H. Zhang Q.-Y. Chen L. Bone marrow mesenchymal stem cells promote osteosarcoma cell proliferation and invasion World J. Surg. Oncol. 13 2015 52 25890096
39 Pan S. Cesarek M. Godoy C. Co C.M. Schindler C. Padilla K. Haskell A. Barreda H. Story C. Poole R. Morpholino-driven blockade of Dkk-1 in osteosarcoma inhibits bone damage and tumour expansion by multiple mechanisms Br. J. Cancer 127 2022 43 55 35277659
40 Yang K. Xie Q. Tang T. Zhao N. Liang J. Shen Y. Li Z. Liu B. Chen J. Cheng W. Astragaloside IV as a novel CXCR4 antagonist alleviates osteoarthritis in the knee of monosodium iodoacetate-induced rats Phytomedicine 108 2023 154506 10.1016/j.phymed.2022.154506 36403512
41 Yang K. Xie Q. Liang J. Shen Y. Li Z. Zhao N. Wu Y. Liu L. Zhang P. Hu C. Identification of Andrographolide as a novel FABP4 inhibitor for osteoarthritis treatment Phytomedicine 118 2023 154939 10.1016/j.phymed.2023.154939 37354697
