Abstract 6923: The EHD2-Orai1 pro-oncogenic axis is required for calcium-dependent membrane repair in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive and highly metastatic breast cancer subtype characterized by poor therapeutic outcomes. Increased invasive capabilities expose tumor cells to higher plasma membrane stress resulting in frequent breaches that are incompatible with cancer cell survival and invasiveness. Upregulation of membrane repair mechanisms has been shown to counteract this inherent tumor cell vulnerability and targeting such mechanisms represents a novel potential therapeutic strategy. Previous studies have shown that plasma membrane repair involves multiple calcium-dependent protein complexes. We have previously demonstrated that EHD2, a member of the EHD (Eps15 homology domain-containing) protein family, functions in this role by sustaining the plasma membrane expression of Orai1, which is required to sustain the pro-oncogenic Store-Operated Calcium Entry (SOCE) pathway. EHD2 has been found to accumulate at plasma membrane repair sites in skeletal muscle models, suggesting the potential role of EHD2-SOCE axis in regulating plasma membrane integrity in breast cancer cells. Methods: We used CRISPR-Cas9 knockout of EHD2 or Orai1 as well as Orai1 inhibition in TNBC cell lines to examine the role of EHD2-Orai1 axis in the repair of mechanical or streptolysin O (SLO) toxin induced plasma membrane disruptions. The role of EHD2 as a mechano-transducer of external plasma membrane stress leading to Orai1 activation was investigated using micro-cantilevers to indent TNBC cells expressing genetic encoded calcium sensors. Functional linkage of EHD2 and Orai1 was assessed by measuring the impact of gene Knockout or complementation on SOCE and cell migration. Results: EHD2 knockout impaired the ability of TNBC cells to efficiently repair the plasma membrane disruptions induced by mechanical injury or SLO. Orai1 knockout recapitulated the EHD2 knockout phenotype. A functional linkage of EHD2 and Orai1 was demonstrated by the ability of ectopic constitutively active Orai1, but not its dominant negative form, to rescue the cell migration defects of EHD2 knockout TNBC cells. Indenting the plasma membrane with micro-cantilevers induced rapid Ca2+ flux in TNBC cells and accumulation of EHD2 at the sites of deformation. Membrane deformation-induced Ca2+ entry was markedly reduced by EHD2 or Orai1 knockout. Orai1 inhibitors impaired the repair of mechanical or SLO induced plasma membrane disruption. Conclusion: Our results reveal an EHD2-Orai1 axis that is a critical regulator of mechanosensitive Ca2+ entry in TNBC cells and required for their plasma membrane repair. Our findings support a role of EHD2-Orai1 axis in mechano-sensing and plasma membrane integrity in TNBC cells under stress. These findings suggest that targeting the EHD2-SOCE axis could present a novel therapeutic strategy in TNBC. Citation Format: Haitao Luan, Azize Cerci, Bhopal Mohapatra, Timothy Bielecki, Matthew Storck, Jun-Yul Lim, Vimla Band, Subu Ramanathan, Hamid Band. The EHD2-Orai1 pro-oncogenic axis is required for calcium-dependent membrane repair in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6923.