AGAP1 and noncanonical GDP binding regulate triple-negative breast cancer development.

AGAP1, a key regulator of the cytoskeleton, plays a vital role in cell migration and invasion. Aberrant expression of AGAP1 is frequently associated with a variety of severe diseases, including triple-negative breast cancer (TNBC). However, the pathogenic mechanism underpinning the AGAP1-driven disease progression is yet to be understood. Here, we observed that genetic knockout of AGAP1 significantly promoted TNBC tumor growth and metastasis. This led to aberrant activation of glycolysis and tricarboxylic acid cycle (TCA) metabolic pathway in the subcutaneous xenograft model and pulmonary metastasis mouse model. Using X-ray crystallography, we determined the crystal structure of the AGAP-GLD domain in complex with GDP nucleotide (2.5 Å), revealing a previously unrecognized noncanonical GDP-binding site delineated by three relatively conserved residues, R106-F107-K108. Structure-based mutagenesis assays showed that the disruption of this GDP-binding site markedly enhanced the proliferation and migration of TNBC cells. RNA-seq transcriptome profiling in cancer cells again highlighted the glycolysis and TCA cycle regulated by AGAP1-GDP interaction. This was further supported by ECAR, OCR and glucose uptake measurements, alongside enhanced PI3K–AKT activation and PI3K dependence, as copanlisib suppressed AGAP1 loss–induced proliferation. Supportively, AGAP1 was downregulated in TNBC patients, associated with an activated immune–stromal microenvironment. The AGAP1 mutations near the GDP-binding site was often correlated with poorer patient survival, consistent with the structure-based animal experiments. Altogether, these results helped to define a previously unrecognized noncanonical GDP-binding site in AGAP1 that might play a critical role in TNBC progression.