The hexosamine biosynthetic pathway alters the cytoskeleton to modulate cell proliferation and migration in aggressive prostate cancer

Castration-resistant prostate cancer (CRPC) progresses despite androgen deprivation therapy, as cancer cells adapt to grow without testosterone, becoming more aggressive and prone to metastasis. CRPC biology complicates the development of effective therapies, posing challenges for patient care. Recent gene-expression and metabolomics studies highlight the Hexosamine Biosynthetic Pathway (HBP) as a critical player, with key components like GNPNAT1 (Glucosamine-phosphate N-acetyltransferase 1) being downregulated in CRPC. GNPNAT1 knockdown in pre-clinical models has been shown to increase growth and metastasis in CRPC tumors, though the mechanisms remain unclear.To investigate the cellular basis of these CRPC phenotypes, we generated a CRISPR-Cas9 knockout model of GNPNAT1 in 22Rv1 CRPC cells, analyzing its impact on transcriptomic and glycoproteomic profiles of cells. We find that HBP inhibition disrupts the cytoskeleton, altering mitotic progression and promoting uncontrolled growth. GNPNAT1 KO cells showed reduced levels of cytoskeletal filaments, such as actin and microtubules, leading to cell structure disorganization and chromosomal mis-segregation. GNPNAT1 inhibition also activated PI3K/AKT signaling, promoting cell proliferation, impaired cell adhesion by mis-localizing Eph Receptor B6, enhancing migration via the RhoA (Ras homolog family member A) pathway and promoting epithelial-to-mesenchymal transition. These findings suggest that HBP plays a critical role in regulating CRPC cell behavior, and targeting this pathway could provide a novel therapeutic approach.