Simple Summary
Triple-negative breast cancer (TNBC) is an aggressive and hard-to-treat form of breast cancer that has limited treatment options. In TNBC, changes in DNA (mutations) associated with the cancer, frequently occur in the TP53 gene. The TP53 gene normally helps cells repair damage or self-destruct when they are not functioning properly; however when mutated, TP53 cannot function properly. In this study, we found that different types of TP53 mutations, affect how cancer cells manage calcium—an important signal that controls how cells grow and die. Some of these mutations made the cancer cells less likely to die under stress, which may explain why they can resist certain treatments. To address this problem, we tested a TP53 reactivator that helps restore the normal functions of TP53. This TP53 reactivator increased calcium levels in the cancer cells and made them more responsive to treatment. Our findings help explain why some cancers are more aggressive and suggest a new approach to treatment by targeting both the mutant TP53 and the disrupted calcium signals. This could lead to better therapies and improved outcomes for people with TNBC.
Abstract
Background: Triple-negative breast cancer (TNBC) is an aggressive subtype lacking estrogen, progesterone, and HER2 receptors, and is associated with poor prognosis and limited targeted therapeutic options. TP53 mutations occur in the majority of TNBC cases, disrupting p53’s role in DNA repair and apoptosis. Beyond gene regulation, p53 also influences calcium signalling through store-operated calcium entry (SOCE), a critical pathway for cell survival and death. However, the impact of different TP53 mutation types on calcium signalling remains unclear. Methods: Calcium channel gene expression was analysed using publicly available TNBC datasets. Calcium channel expression and SOCE activity were assessed in TNBC cell lines with different TP53 mutations using quantitative PCR and calcium imaging (Fura-2AM). Cell proliferation was measured using acid phosphatase assays, while apoptosis was evaluated through caspase 3/7 activation using the Incucyte live-cell fluorescent imager. The p53 reactivator COTI-2 was tested for its ability to restore TP53 function and modulate calcium signalling. Results: Analysis revealed significant downregulation of CACNA1D in TP53-mutant TNBCs. TNBC cell lines harbouring frameshift and stop TP53 mutations exhibited reduced SOCE, lower CACNA1D expression, and resistance to thapsigargin-induced apoptosis compared to wild-type cells. In contrast, cells with the TP53 R273H missense mutation demonstrated similar calcium signalling and proliferation to TP53 wild-type cels. COTI-2 treatment restored CACNA1D expression and SOCE in frameshift and stop mutant cells, enhancing apoptotic sensitivity. Combined treatment with COTI-2 and thapsigargin resulted in a synergistic increase in apoptosis. Conclusions: This study identifies a novel link between TP53 mutation type and calcium signalling in TNBC. Reactivating mutant p53 with COTI-2 restores calcium-mediated apoptosis, supporting combination strategies targeting both TP53 dysfunction and calcium signalling.