Mitochondrial Localization of Cx26 Promotes Colorectal Cancer Malignancy and Exposes a Fatal Vulnerability to Drug Targeting

ABSTRACT The malignant progression of colorectal cancer (CRC) is closely linked to metastasis and therapy resistance, but underlying mechanisms remain unclear. Here, we identify mitochondrial translocation of connexin 26 (Cx26) as a hallmark of highly malignant metastatic CRC and reveal its critical role in promoting ER‐mitochondria crosstalk, calcium signaling, and epithelial‐mesenchymal transition (EMT). Mechanistically, we demonstrate that protein L‐isoaspartyl/D‐aspartyl O‐methyltransferase (PIMT) methylates Cx26 at asparagine 176, enhancing its mitochondrial localization and stability. This modification disrupts Cx26's gap junction function while activating calcium signaling to drive EMT and metastasis. Furthermore, we discovered that bortezomib (BTZ), contrary to its canonical proteasome inhibition mechanism, exhibits exceptional efficacy against highly malignant CRC by targeting the PIMT‐Cx26 axis. BTZ directly binds PIMT, further amplifying Cx26 mitochondrial translocation and hyperactivating Ca 2 + ‐ROS‐ERK signaling, ultimately inducing lethal autophagy and apoptosis in metastatic CRC cells. Strikingly, low‐malignancy primary CRC cells remain resistant to BTZ, highlighting its selectivity for advanced disease. Collectively, our study uncovers mitochondrial Cx26 as a novel therapeutic vulnerability in metastatic CRC and repurposes BTZ as a precision strategy targeting PIMT.