Both protein kinase C (PKC) and reactive oxygen species (ROS) are well-known signaling messengers cross-talking with each other to activate mitogen-activated protein kinases (MAPKs) for progression of hepatocellular carcinoma (HCC). However, the underlying mechanisms are not well elucidated. Especially, whether mitochondrial ROS (mtROS) is involved and how it triggers MAPK signaling are intriguing. In this study, we found mtROS generation and phosphorylation of MAPKs were mediated by PKCδ in HCCs treated with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Heat shock protein 60 (HSP60), one of the chaperones in mitochondria was the major protein oxidized in TPA-treated HCCs. Moreover, depletion of HSP60 or expression of HSP60 cysteine mutant prevented TPA-induced phosphorylation of MAPKs. To delineate how HSP60 mediated MAPK activation, the role of Raf kinase inhibitor protein (RKIP), a negative regulator of MAPK, was investigated. TPA dissociated RKIP from HSP60 in both mitochondria and cytosol, concurrently with translocation of HSP60 and MAPK from mitochondria to cytosol, which was associated with robust phosphorylation of MAPKs in the cytosol. Moreover, TPA induced opposite phenotypical changes of HCCs, G1 cell cycle arrest, and cell migration, which were prevented by mtROS scavengers and depletion of PKCδ and HSP60. Consistently, TPA increased the migration-related genes, hydrogen peroxide inducible clone5, matrix metalloproteinase-1/3, lamininγ2, and suppressed the cell cycle regulator cyclin E1 (CCNE1) via PKCδ/mtROS/HSP60/MAPK-axis. Finally, c-jun and c-fos were required for TPA-induced expression of the migration-related genes and a novel microRNA, miR-6134, was responsible for TPA-induced suppression of CCNE1. In conclusion, PKCδ cross-talked with mtROS to trigger HSP60 oxidation for release of RKIP to activate MAPK, regulating gene expression for migration, and G1 cell cycle arrest in HCC. Targeted therapy aiming at key players like PKCδ, RKIP, and HSP60 is promising for preventing HCC progression.

Targeting multiple signaling pathways has been proposed as a strategy to overcome resistance to single-pathway inhibition in cancer therapy. A previous study in epithelial ovarian cancers identified hyperactivity of spleen tyrosine kinase (SYK) and EGFR, which mutually phosphorylate and activate each other. Given the potential for pharmacologic inhibition of both kinases with clinically available agents, this study aimed to assess the antitumor efficacy of both pharmacologic and genetic SYK and EGFR coinhibition using a multifaceted approach. We assessed the coinactivation effects in chemoresistant ovarian cancer cell lines, patient-derived organoids, and xenograft models. Dual inhibition of SYK and EGFR in chemoresistant ovarian cancer cells elicited a synergistic antitumor effect. Notably, the combined inhibition activated the DNA damage response, induced G1 cell-cycle arrest, and promoted apoptosis. The phosphoproteomic analysis revealed that perturbation of SYK and EGFR signaling induced a significant reduction in both phosphorylated and total protein levels of cell division cycle 6, a crucial initiator of DNA replication. Together, this study provides preclinical evidence supporting dual inhibition of SYK and EGFR as a promising treatment for chemoresistant ovarian cancer by disrupting DNA synthesis and impairing formation of the prereplication complex. These findings warrant further clinical investigation to explore the potential of this combination therapy in overcoming drug resistance and improving patient outcomes. Significance: SYK and EGFR coinhibition exerts synergistic anticancer effects in chemoresistant ovarian cancer, providing a strategy to treat chemotherapy-resistant ovarian cancers using clinically available agents by targeting critical signaling pathways involved in DNA replication.

Effect of R406 or lapatinib in drug-resistant cell lines

Effects of R406 and lapatinib on cell cycle