Target actin-microtubule network integrity to enhance taxane drug-target engagement and chemosensitivity in prostate cancer

Taxanes exert anticancer activity by binding beta-tubulin on the luminal surface of microtubules, stabilizing protofilaments, suppressing GTP hydrolysis, and thereby promoting drug-target engagement (DTE) and apoptotic cell death. Access of taxanes to luminal binding sites depends on protofilament organization and microtubule lattice integrity, including entry via plus-end openings, small lattice pores, and stress-induced lattice defects. Because microtubules are tightly coupled to the actin cytoskeleton through crosslinking and mechanical support, we hypothesized that kinases controlling actin-microtubule architecture modulate taxane DTE and sensitivity. We investigated the PAK6-LIMK1 signaling axis in human prostate cancer PC3 and DU145 cells using CRISPR/Cas9 PAK6 knockout and small-molecule inhibitors of PAK6 or LIMK1. Taxane chemosensitivity was quantified by cell viability assays, and cytoskeletal organization and DTE were assessed by immunofluorescence microscopy of F-actin, alpha-tubulin, and the +TIP protein CLIP170. Consistent with prior shRNA data, genetic ablation of PAK6 markedly increased taxane sensitivity in both cell lines. PAK6 loss or pharmacologic inhibition produced concordant, distinctive remodeling of the actin-microtubule network, characterized by loss of dorsal, ventral, and transverse stress fibers and the emergence of crooked, banded microtubule cables, indicating compromised cytoskeletal integrity. These structural changes were accompanied by enhanced taxane-induced microtubule bundling and a robust increase in CLIP170 comet number and size, identifying a novel cytoskeletal signature associated with heightened taxane responsiveness. Similar phenotypes and chemosensitization were observed following LIMK1 inhibition, supporting a shared PAK6-LIMK1 pathway that dually regulates actin and microtubule dynamics. Together, these data suggest that disruption of PAK6-LIMK1 signaling weakens cytoskeletal resilience, facilitates taxane access to intraluminal binding sites, and thereby amplifies DTE and cell killing in prostate cancer cells, nominating this axis as a tractable target to overcome taxane resistance in advanced disease.