Comprehensive Proteomics Reveals Pachymic Acid Targeting RBMX via hnRNPC Degradation in Colorectal Cancer

Pachymic acid (PA), a major bioactive component of Poria cocos, suppresses colorectal cancer (CRC) proliferation and invasion by disrupting glycolysis. However, its antitumor activity and underlying mechanisms in vitro and in vivo remain unclear. RNA-binding proteins (RBPs) are key post-transcriptional regulators in CRC, but their mechanisms of action and therapeutic potential remain poorly understood. Here, we employed limited proteolysis-mass spectrometry (LiP-MS), a target deconvolution approach that does not require prior chemical modification, to profile changes in protein conformation within complex proteomes upon PA treatment in cells. Our findings revealed that RBMX (RNA-binding motif protein X-linked) acts as a target of PA and undergoes concentration-dependent conformational changes, as supported by binding validation and functional assays performed in vitro and in vivo. Furthermore, proximity-labeling mass spectrometry (PL-MS) analysis revealed that RBMX interacts with heterogeneous nuclear ribonucleoprotein C (hnRNPC). Functional assays demonstrated that PA treatment induces RBMX-dependent degradation of hnRNPC, thereby suppressing CRC cell proliferation and tumor growth. In summary, this study identifies RBMX as a molecular target of PA through LiP-MS, providing new insights into the mechanisms underlying PA-mediated CRC suppression and highlighting the RBMX–hnRNPC axis as an important mediator of its antitumor effects.