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.
背景：三阴性乳腺癌（TNBC）是一种侵袭性亚型，缺乏雌激素、孕激素和HER2受体，预后较差且靶向治疗选择有限。TP53突变在大多数TNBC病例中发生，破坏了p53在DNA修复和细胞凋亡中的作用。除基因调控外，p53还通过钙库操纵性钙内流（SOCE）影响钙信号传导，这是细胞存活与死亡的关键通路。然而，不同TP53突变类型对钙信号的影响尚不明确。 方法：利用公开的TNBC数据集分析钙通道基因表达。通过定量PCR和钙成像技术（Fura-2AM）评估携带不同TP53突变的TNBC细胞系中钙通道表达及SOCE活性。采用酸性磷酸酶法检测细胞增殖，通过Incucyte活细胞荧光成像仪检测caspase 3/7活性评估细胞凋亡。测试p53再激活剂COTI-2恢复TP53功能及调节钙信号的能力。 结果：分析显示TP53突变型TNBC中CACNA1D显著下调。与野生型细胞相比，携带移码和终止突变的TNBC细胞系表现出SOCE降低、CACNA1D表达减少，并对毒胡萝卜素诱导的凋亡产生抵抗。而携带TP53 R273H错义突变的细胞则表现出与TP53野生型细胞相似的钙信号传导和增殖特性。COTI-2处理可恢复移码和终止突变细胞的CACNA1D表达及SOCE，增强其凋亡敏感性。COTI-2与毒胡萝卜素联合治疗可协同促进细胞凋亡。 结论：本研究揭示了TNBC中TP53突变类型与钙信号传导之间的新联系。使用COTI-2再激活突变型p53可恢复钙介导的细胞凋亡，这为同时靶向TP53功能障碍和钙信号通路的联合治疗策略提供了理论支持。
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