High RSPO3 protein expression serves as an independent poor prognostic factor and promotes malignant progression in...

To elucidate the expression pattern, clinical prognostic significance, and distribution characteristics of R-spondin 3 (RSPO3) within the tumor microenvironment (TME) of breast cancer (BC), to observe its effects on the malignant biological behaviors of MCF-7 cells, and to explore the possible mechanisms underlying its function.

Integrated mRNA expression analysis was performed across multiple cohorts using The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases and validated by quantitative reverse transcription polymerase chain reaction (qRT PCR) in clinical samples. Immunohistochemistry (IHC) was used to detect RSPO3 protein expression in breast cancer tissue microarrays (TMAs), and the results were combined with survival data to assess prognostic value. Single‑cell transcriptomics data were interrogated to identify the primary cellular sources of RSPO3, followed by differential gene expression and pathway enrichment analyses in key subpopulations. Finally, in vitro functional assays were conducted to investigate the effects of RSPO3 on breast cancer cell proliferation, migration, and apoptosis. Specifically, a co‑culture system of human breast cancer‑associated fibroblasts (HBCFs) and MCF‑7 cells was employed to examine changes in RSPO3 expression and the proliferative and migratory capacities of breast cancer cells after co‑culture. The function of RSPO3 was verified by knocking down RSPO3 in the co‑culture system, and further validation was carried out through Western blotting, CHIR99021 activator rescue experiments, and tissue analysis.

Compared to adjacent non-tumor tissues, RSPO3 mRNA expression was generally downregulated in BC tissues; however, RSPO3 protein levels were significantly higher in tumor tissues. High RSPO3 protein expression was an independent risk factor for reduced overall survival (OS). Single-cell analysis revealed that within the TME, RSPO3 was primarily expressed in fibroblasts, endothelial cells, and epithelial cells. Notably, among fibroblasts, RSPO3 expression was specifically enriched in the inflammatory cancer-associated fibroblast (iCAF) subpopulation. RSPO3⁺ iCAFs exhibited functional characteristics related to cell proliferation regulation, angiogenesis, Wnt signaling, and oxidative phosphorylation. In vitro experiments demonstrated that RSPO3 knockdown suppressed the proliferation, migration, and invasion of MCF-7 cells while promoting apoptosis.Furthermore, co-culture of HBCFs with MCF-7 cells significantly increased RSPO3 protein levels in MCF-7 cells, accompanied by markedly enhanced proliferation and migration abilities. Knockdown of RSPO3 significantly reversed these pro-tumorigenic effects. RSPO3 knockdown downregulated the expression of Axin2, Cyclin D1, and β-catenin. Treatment with the Wnt pathway activator CHIR99021 partially rescued the inhibition of invasion and migration induced by RSPO3 knockdown. Immunohistochemical staining of breast cancer tissues further confirmed the positive correlation between RSPO3 expression and β-catenin and Cyclin D1 expression.

RSPO3 exhibited significant discrepancies between transcriptional and translational levels in breast cancer tissues. High RSPO3 protein expression indicated poor prognosis and served as an independent prognostic risk factor. Breast cancer-associated fibroblasts may promote tumor cell proliferation and migration by regulating RSPO3 expression and subsequently activating the Wnt/β-catenin pathway. RSPO3 represents a potential prognostic biomarker and therapeutic target.