First functional evidence that a rare germline TP53β variant drives senescence-associated immune suppression and impairs apoptosis and cell migration in breast cancer patients

INTRODUCTION: Pathology-supported genetic testing (PSGT), a personalized medicine framework established in South Africa, led to the identification of a rare germline tumour suppressor protein 53 (TP53) beta-isoform (β) variant (NM_001126114.3, c.1018A>G, p.N340D) in a family with the Li-Fraumeni-like syndrome. While protein modeling predicted structural alterations consistent with impaired function, its pathogenicity remained unclear. AIM: To determine the functional impact of the TP53β N340D variant on cell proliferation, cell death, senescence, migration, and anti-tumour activity using a translational ex vivo model. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from female controls and breast cancer patients, with or without the TP53β N340D variant. Lipopolysaccharide (LPS)- and phytohemagglutinin-L (PHA-L)-stimulated proliferation was evaluated by a water-soluble tetrazolium 1 (WST-1) assay. Doxorubicin (DXR)-induced cell death was assessed using a WST-1 assay, flow cytometry, and western blotting. A senescence-associated beta-galactosidase assay and western blotting determined senescence. Migration and anti-tumour activity were assessed using a Transwell assay and co-culturing PBMCs with BT-549 spheroids. RESULTS: The TP53β N340D variant impaired DXR-induced cell death (p < 0.001), supported by reduced late apoptosis and decreased CASP3 and PARP activation. TP53β N340D PBMCs exhibited increased senescence (p < 0.01), potentially contributing to chemoresistance. Reduced LPS- and PHA-L-stimulated proliferation was dependent on cancer status. The variant reduced PBMC migration (p < 0.01), suggesting altered immune recruitment. Although anti-tumour activity appeared reduced in TP53β N340D PBMCs, spheroid size remained unchanged. CONCLUSIONS: This study provides supporting evidence for the pathogenicity of the TP53β N340D variant and highlights the importance of integrating functional genomics into PSGT to enhance medical decision-making.