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Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor (LATS) Biosensor
Published on: September 13, 2018
Targeting the Hippo/YAP-TAZ pathway increases X-ray sensitivity in chondrosarcoma spheroids and is associated with
Birgit Lohberger1, Dietmar Glänzer1, Heike Kaltenegger1
1Department of Orthopedics and Trauma, Medical University of Graz, Auenbruggerplatz 5, 8036 Graz, Austria.
Background:
Chondrosarcomas are malignant cartilage-forming bone tumors with limited therapeutic options, as surgical resection remains the only curative treatment, while chemo- and radiotherapy show limited efficacy due to intrinsic resistance mechanisms. Dysregulation of the Hippo signaling pathway been associated with tumor progression and therapy resistance and has emerged as a promising experimental target across several cancer entities. However, its relevance in chondrosarcoma remains unclear, particularly with regard to potential radiosensitizing effects.
Methods:
Human chondrosarcoma (SW-1353 and Cal78) and healthy chondrocyte (HC) spheroids were treated with Verteporfin (VP) alone or in combination with X-ray irradiation. Cell viability, Hippo/YAP-TAZ signaling, autophagy-associated markers, DNA damage, apoptosis, ultrastructural alterations, and gene expression were analyzed using ATP assays, immunoblotting, RT-qPCR, immunohistochemistry, and transmission electron microscopy.
Results:
VP treatment caused a dose-dependent decline in cell proliferation and suppressed key YAP/TAZ target genes, both in chondrosarcoma cells and HC, alongside downregulation of Hippo pathway components and proliferative markers like cMyc and PCNA. Combined with X-ray irradiation, these effects were amplified, supporting a potential radiosensitizing activity of VP in this preclinical model. Ultrastructural and molecular data showed that VP disrupted autophagic balance in all cell types, leading to mitochondrial damage, lipid accumulation, and nuclear fragmentation. Despite the higher basal autophagy in HC cells, VP led to cell death involving autophagy, apoptosis, and DNA damage, evidenced by increased LC3B-II, Beclin, γH2AX, and PARP cleavage. Enhanced DNA damage markers and altered p53-MDM2 signaling after treatment reflect the genotoxic stress induced.
Conclusion:
Our data suggest that VP, particularly in combination with irradiation, is associated with disruption of multiple survival pathways in chondrosarcoma cells, offering a promising avenue for therapeutic intervention. Future studies should investigate the interplay between autophagy, apoptosis, and Hippo signaling to enable their therapeutic targeting in cancer.
Insights
Verteporfin (VP) combined with irradiation shows promise for treating chondrosarcoma by disrupting cell survival pathways. This approach targets Hippo signaling and induces cell death, offering a potential new therapy for this bone cancer.
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- Chondrosarcomas are malignant bone tumors with limited treatment options, often resistant to chemotherapy and radiotherapy.
- The Hippo signaling pathway is implicated in tumor progression and therapy resistance but its role in chondrosarcoma is not well understood.
- Investigating the Hippo pathway's relevance in chondrosarcoma could reveal new therapeutic strategies, particularly for radiosensitization.
Purpose of the Study:
- To investigate the effect of Verteporfin (VP), a Hippo pathway inhibitor, on chondrosarcoma cells.
- To evaluate the potential radiosensitizing effect of VP in combination with X-ray irradiation on chondrosarcoma.
- To explore the underlying mechanisms, including effects on cell viability, signaling pathways, autophagy, and DNA damage.
Main Methods:
- Human chondrosarcoma cell lines (SW-1353, Cal78) and healthy chondrocytes were cultured as spheroids.
- Cells were treated with VP alone or in combination with X-ray irradiation.
- Analyses included cell viability assays, Western blotting, RT-qPCR, immunohistochemistry, and transmission electron microscopy to assess signaling, autophagy, DNA damage, and apoptosis.
Main Results:
- VP treatment reduced chondrosarcoma cell proliferation and suppressed YAP/TAZ target genes, downregulating Hippo pathway components.
- Combining VP with X-ray irradiation amplified these effects, indicating radiosensitizing potential.
- VP disrupted autophagic balance, induced mitochondrial damage, and triggered cell death via autophagy, apoptosis, and DNA damage, evidenced by molecular markers.
Conclusions:
- Verteporfin, especially with irradiation, disrupts multiple survival pathways in chondrosarcoma cells, presenting a promising therapeutic strategy.
- Further research into the interplay between autophagy, apoptosis, and Hippo signaling is crucial for developing targeted cancer therapies.
- This study provides a preclinical basis for exploring VP as a radiosensitizer in chondrosarcoma treatment.