SPDEF downregulation promotes tumor aggressiveness and poor prognosis in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype associated with high rates of recurrence, metastasis, and poor prognosis. Identifying key molecular regulators is essential for improving prognostic assessment and therapeutic strategies in TNBC. However, the expression pattern, clinical significance, and underlying regulatory mechanisms of SAM pointed domain-containing ETS transcription factor (SPDEF) in TNBC remain unclear. This study aims to investigate the expression pattern, clinical significance, and underlying regulatory mechanisms of SPDEF in regulating tumor cell biological behaviors in TNBC.

Immunohistochemical staining was performed to assess SPDEF expression across different breast cancer subtypes in samples collected at our center. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analyses were used to determine SPDEF expression levels in normal mammary epithelial cells and various breast cancer cell lines. SPDEF overexpression and knockdown models were established in MDA-MB-468 and MDA-MB-231 TNBC cells, respectively. Cell proliferation, migration, invasion, and apoptosis were evaluated using Cell Counting Kit-8 (CCK-8), colony formation, wound healing, Transwell invasion assays, and flow cytometry. Apoptosis-related protein expression was examined by Western blotting, and spatial transcriptomic data were integrated to analyze the spatial association between SPDEF and apoptosis-related genes.

SPDEF expression was markedly reduced in TNBC tissues and cell lines, whereas significantly higher expression was observed in human epidermal growth factor receptor 2 (HER2) positive, luminal A, and luminal B breast cancer subtypes. Clinically, low SPDEF expression was significantly associated with advanced tumor-node-metastasis (TNM) stage and tumor recurrence in patients with TNBC. Survival analysis demonstrated that patients with high SPDEF expression exhibited significantly improved recurrence-free survival and overall survival compared with those with low SPDEF expression. Functional assays revealed that SPDEF knockdown significantly enhanced TNBC cell proliferation, migration, and invasion while suppressing apoptosis, whereas SPDEF overexpression exerted opposite effects. Mechanistically, SPDEF promoted apoptosis by upregulating pro-apoptotic proteins Bax, cleaved caspase-3, and cleaved caspase-9, while downregulating anti-apoptotic proteins Bcl-2 and Bcl-xL. Spatial transcriptomic analysis further confirmed significant enrichment of apoptosis-related genes CASP3 and CASP9 in SPDEF-low expression regions of TNBC tissues.

SPDEF is specifically downregulated in TNBC, and its high expression is associated with earlier tumor stage and favorable clinical outcomes. SPDEF suppresses TNBC cell proliferation, migration, and invasion while promoting apoptosis through modulation of apoptosis-related protein expression, highlighting its potential as a prognostic biomarker and therapeutic target for TNBC.