WNT Pathway Molecular Alterations: From Biomarkers to Therapeutic Opportunities in Soft Tissue Sarcomas

eng Introduction: Soft tissue sarcomas (STS) are a rare and heterogeneous group of malignancies arising from mesenchymal tissues, encompassing over 100 distinct histological subtypes. Despite representing less than 1% of all adult cancers, STS poses a substantial clinical challenge due to its aggressive behavior, high recurrence rates, and limited therapeutic options. Surgery remains the primary treatment modality for localized disease, often in combination with radiotherapy and/or chemotherapy. In the advanced setting of STS, where the median survival remains approximately 12 months, doxorubicin has been the gold standard treatment for the past forty years. Given the inherent heterogeneity, genomic complexity, and molecular diversity of STS, there is an urgent need for novel, more effective, and personalized therapeutic strategies. Recent advances in cancer research have implicated the WNT signaling pathway, an evolutionarily conserved pathway critical for cellular processes such as proliferation, differentiation, and migration, in the pathogenesis of many cancers, including sarcomas. Previous work by our research group demonstrated the aberrant activation of the WNT signaling pathway in STS, positioning it as a potential therapeutic target. This doctoral Thesis explores the role of the WNT signaling pathway in STS, with the overarching objective of characterizing its molecular alterations, assessing the therapeutic potential of WNT inhibitors, and identifying actionable biomarkers to improve patient outcomes. Research content: The research employed innovative methodologies, including patient-derived primary cultures and three-dimensional organoids, as well as liquid biopsy for biomarker discovery. Comprehensive molecular analyses revealed heterogeneous activation of the canonical WNT/β-catenin signaling pathway in STS, with elevated nuclear β-catenin expression and transcriptional activity detected mainly in undifferentiated sarcoma-derived primary cultures. Next-generation sequencing identified actionable genetic alterations in WNT pathway regulators in 37% of STS tumors analyzed, including pathogenic variants in APC and SOX9, along with mutations in the DNA damage and PI3K/AKT/mTOR pathways. Downstream inhibition of the WNT signaling pathway significantly reduced cell viability in sarcoma-derived cell culture models. In particular, the small molecule SM08502, which targets β-catenin-mediated transcription and is being evaluated in clinical trials for other malignancies, emerged as the most effective targeted therapy. The sensitivity of STS primary cultures to WNT inhibitors correlated with WNT transcriptional activation, and nuclear β-catenin, WNT5B expression levels and histological grade of STS tumors were identified as predictive biomarkers of response to SM08502. Furthermore, undifferentiated sarcomas exhibited superior responses to WNT signaling inhibition than the other studied STS subtypes. WNT5A and WNT5B ligands were identified as key modulators of STS progression, with elevated expression levels associated with poor overall survival and earlier recurrence. Elevated WNT5A levels in plasma-derived extracellular vesicles from STS patients were significantly associated with recurrence, suggesting its potential for disease monitoring and early detection of relapse in STS. Functional studies demonstrated the impact of WNT5A downregulation on STS cell migration and epithelial-mesenchymal transition (EMT), which are critical processes for tumor invasion and metastasis. Both the inhibition of WNT5A expression and the treatment with the WNT5A antagonist Box5 led to a reduction in the migration capacity of 75% of STS in vitro models, with a greater effect observed following WNT5A silencing. These findings highlight the translational potential of WNT5A as a non-invasive prognostic biomarker and a potential therapeutic strategy to limit STS progression. Moreover, a composite biomarker combining WNT5A and WNT5B expression enhanced the prognostic stratification of STS patients, providing a more accurate tool for assessing survival and recurrence risks. Conclusion: The integration of state-of-the-art methodologies in this study has led to significant advancements in the understanding of WNT signaling in STS and its therapeutic implications. The establishment of robust preclinical models and the identification of clinically relevant biomarkers open avenues for personalized medicine approaches. This work represents a pivotal contribution to the field, offering a foundation for improved prognostic and therapeutic paradigms in this challenging and diverse group of malignancies.