Abstract PO2-24-08: GD3 Synthase is a Master Regulator of Wild-Type p53–Mediated Apoptosis and Mutant p53–Mediated Tumorigenesis
Authors: Vivek Anand, FOUAD EL-DANA, JENNY BORGMAN, MICHAEL ANDREEFF, Venkata Lokesh Battula
Journal: Cancer Research (2024)

Abstract The tumor suppressor protein p53 is essential for maintaining genomic stability and regulating cellular responses to stress. TP53 mutations are common in breast cancer (BC) and other malignancies. We previously showed that ganglioside GD2 identifies BC stem-like cells (BCSCs) and that GD3 synthase (GD3S/ST8SIA1) is upregulated in breast tumors with TP53 mutations. Here, we demonstrate the direct involvement of p53 in the transcriptional regulation of GD3S in the GD2 biosynthesis pathway. Our results uncover a novel role of GD3S in inhibiting p53-mediated apoptosis and promoting mutant p53–induced tumor initiation. To investigate the p53-mediated regulation of GD3S expression, we used the TCGA and METABRIC datasets and examined GD3S mRNA expression in breast tumors with different p53 mutation status. GD3S expression was significantly upregulated in BC patients with hotspot (HS) p53 mutations compared to those with wild-type (WT) p53 or non-HS p53 mutations (P&amp;lt; 0.0001). Immuno-histochemical analysis of GD3S and p53 in FFPE primary tumor tissues from BC patients (n=84) with varying p53 expression status demonstrated that patients with HS p53 mutations had higher histological scores for GD3S than did patients with WT p53 (P&amp;lt; 0.0001) or non-HS p53 mutations (P=0.009). GD3S and p53 expression were significantly positively correlated in patients with HS p53 mutations (r=0.735; P=0.006) but not in patients with WT p53 (r=–0.09) or non-HS p53 mutations (r=0.14). Consistent with these patient data, BC cells harboring HS p53 mutations had significantly upregulated and positively correlated expressions of GD3S and p53 (n=8; r=0.85). Moreover, compared to those with WT p53, the cells with HS p53 mutations had substantially greater populations of GD2+ BCSCs (P=0.02) and GD3+ BCSCs (P=0.01). Subsequently, we investigated the regulatory role of p53 in the GD2 biosynthetic pathway by stabilizing WT p53 expression by nutlin-3a treatment in 4 BC cell lines and suppressing the expression of HS p53 mutants by p53 knockdown in their respective cell lines (n=4). In cell lines with WT p53, nutlin-3a significantly decreased GD3S expression and GD2 levels (P&amp;lt; 0.05 and P&amp;lt; 0.0001, respectively), suggesting that WT p53 inhibits GD3S expression. In cell lines with p53 mutations, p53 knockdown significantly inhibited GD3S expression. In both cells with WT p53 (MCF7 and ZR751) and cells with HS p53 mutations (Hs578T and BT549), the stable overexpression of GD3S effectively suppressed apoptosis even when WT p53 was stabilized or HS p53 mutant expression was depleted. Consistent with these results, in mice bearing MCF7 cell–derived xenografts, the overexpression of GD3S counteracted the effect of stabilized WT p53–mediated apoptosis and facilitated tumor growth (P&amp;lt; 0.0001). The results of a promoter-luciferase reporter assay demonstrated that nutlin-3a reduced GD3S promoter activity in a dose-dependent manner in MCF7 and ZR751 cells (P&amp;lt; 0.0001), whereas doxycycline (1µM) significantly reduced GD3S promoter activity in Hs578T and BT549 cells with inducible p53 knockdown (P&amp;lt; 0.001). ChIP-qPCR analysis confirmed specific enrichment of WT p53 at the 300-bp upstream regions and additional enrichment of HS p53 mutants at the downstream regions of the GD3S promoter, indicating that WT p53 and HS p53 mutations have distinct promoter regulation patterns. In summary, our study reveals that GD3S has a previously unknown anti-apoptotic function in BC, in that it helps attenuate p53-mediated apoptosis while activating tumor-promoting pathways that operate independently of p53. Citation Format: Vivek Anand, FOUAD EL-DANA, JENNY BORGMAN, MICHAEL ANDREEFF, Venkata Lokesh Battula. GD3 Synthase is a Master Regulator of Wild-Type p53–Mediated Apoptosis and Mutant p53–Mediated Tumorigenesis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-08.