BSBM-02 Sirpα Intracellular Signaling Mediated Mitochondrial Fission in Triple-Negative Breast Cancer Cells Is Associated With Breast-to-Brain Metastasis

Abstract Triple-negative breast cancer (TNBC), the most aggressive breast cancer subtype, often leads to brain metastasis (35%). Current need for standard treatment requires the development of new therapies. The CD47/SIRPα interaction drives immune escape. However, SIRPα’s expression and role in cancer cells remain poorly understood. Single-cell RNA-Seq data reveals SIRPα upregulation in TNBC, supported by strong immunoreactivity in patient biopsies and a 3.5-fold increase in breast-to-brain metastatic lesions. (n=9-19; p0.01). Consistently, the protein expression of SIRPα TNBC 4T1 and EO771 brain-tropic cells (4T1Br3/EO771Br4) is significantly higher than the parental cells (n=3; p0.05). Stably overexpressed SIRPα and knockout TNBC cell lines show that SIRPα regulates SHP2 phosphorylation in cancer cells, and SIRPα/SHP2 axis regulates TNBC cell migration. According to the gene ontology from Bulk RNA-Seq data on orthotopic tumors, anti-SIRPα treatment affects the genes related to mitochondria and organelle membrane clusters, correlating to mitochondrial fission and fusion dynamics. Our data shows a doubling in Drp1 (mitochondria fission) protein expression in TNBC brain-tropic cells compared to parental, whereas MFN2 (mitochondria fusion) has a 50% decrease in expression. Moreover, SIRPα knockout cell line also downregulates mitochondria fission with decreased Drp1 and increased MFN2 expression. Allosteric SHP2 inhibitor treatment on TNBC brain-tropic cells also shows a dose-dependent inhibition of Drp1 (n=3, p0.001) and MFN2 enhancement (n=3, p=0.0184) with Erk signaling inhibition. SIRPα/SHP2/Erk axis shifts mitochondria fusion to fission, leading to cancer metastasis. SIRPα blockade by antibody reduced metastatic brain lesion formation in two in vivo models by approximately 90% (n=4-7; p 0.05). Spatial proteomics revealed changes in adaptive and innate suppressive and inflammatory signaling that may be associated with the observed changes in SIRPα intracellular signaling. This was associated with shift in ECM proteins that may influence mitochondrial function to influence metastasis., thus providing a potential mechanism for how SIRPα promotes TNBC brain metastasis.