Colorectal cancer (CRC) ranks second in cancer-related mortality worldwide. Its clinical intractability and chemoresistance pose significant challenges, often driven by cancer stem cells (CSCs). Wnt/β-catenin signaling is a lynchpin of CSC self-renewal, but targetable upstream drivers remain elusive. Using bioinformatics, we identified the proteoglycan SPOCK1 (encoding the protein Testican-1) as a Wnt-associated marker. High SPOCK1 gene expression predicted poor patient prognosis and reduced progression-free survival following first-line chemotherapy. In vitro assays demonstrated that Testican-1 levels modulated Wnt/β-catenin activity. Testican-1 downregulation inhibited β-catenin nuclear translocation and TCF/LEF-mediated transcription, leading to concurrent suppression of CRC proliferation and spheroid-forming potential. Conversely, Testican-1 overexpression promoted β-catenin nuclear translocation and TCF/LEF-mediated transcription, which enhanced CRC malignant properties. This Testican-1-driven pro-tumorigenic effect was reversed by the β-catenin/TCF/LEF inhibitor iCRT3, demonstrating Testican-1’s dependence on Wnt transcriptional activation. We validated this functional role in vivo using a Testican-1-overexpressing xenograft model and ex vivo using patient-derived organoids (PDOs), both confirming increased growth and β-catenin nuclear localization. Furthermore, data-independent acquisition (DIA) mass spectrometry of Testican-1-overexpressing tumors showed enrichment in Wnt/β-catenin and pro-tumorigenic pathways, including c-Myc and oxidative phosphorylation. Notably, Testican-1 expression was associated with remodeling of the tumor microenvironment, characterized by desmoplastic features. In summary, our findings establish a novel Testican-1/Wnt/β-catenin axis driving CRC tumorigenesis and progression, positioning it as a promising therapeutic target to overcome Wnt-mediated progression and resistance.
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