Delivery of Pleckstrin‐Homology Domains Suppresses PI3K/Akt Signaling and Breast Cancer Metastasis

ABSTRACT Current cancer therapies inhibit tumor growth but fail to target metastatic dissemination. Obscurin (720–870 kDa), a giant signaling protein localizing to the breast epithelial cell membrane, is a metastasis suppressor commonly lost in breast cancer. Obscurin loss upregulates the oncogenic PI3K/Akt axis. While restoring obscurin expression is crucial from a translational standpoint, it poses major challenges due to its immense size. Herein, we overcome this hurdle by delivering a mini‐obscurin—comprising the obscurin‐pleckstrin homology (PH) domain, which is ∼50‐times smaller than the full‐length protein—into aggressive breast cancer cells via adenovirus and lipid nanoparticles. Mechanistically, the obscurin‐PH‐domain interacts with the PI3K‐p85 regulatory subunit. Membrane‐targeted obscurin‐PH sequesters p85, suppressing PI3K/Akt activity. p85‐sequestration eliminates filopodia, hampering migration and adhesion to pre‐metastatic niche extracellular matrix substrates. This intervention further eradicates invadopodia and reduces matrix metalloproteinase expression, blocking invasion, dissemination, and metastasis. We recapitulate this phenotype using the structurally homologous kalirin and PLCγ1 PH‐domains and ultimately uncover a family of nine PH‐domains that may act as PI3K inhibitors, unified by the “ p 85 i nhibitory m etastasis s uppressor” (PIMS) motif, mediating this effect. This work engineers a first‐in‐class group of non‐chemical PI3K inhibitors, uniquely targeting the PI3K‐p85 subunit, galvanizing novel gene therapies for treating metastatic breast cancer.