ICA-1S targets protein kinase C-iota to inhibit the WNT/beta-catenin signaling in hepatocellular carcinoma

Liver cancer is one of the leading causes of cancer-related mortality in the United States and a significant threat to public health. Despite the projection of the World Health Organization that the global deaths from liver cancer would exceed one million by 2030, liver cancer continues to receive limited attention in oncology drug development. The activation of the WNT/beta-catenin signaling pathway is a key molecular event in hepatocellular carcinoma (HCC), and the canonical pathway is activated in approximately one-third of HCC cases. However, therapeutic targeting of this pathway has been limited by challenges in specificity. In this study, we investigated the role of atypical protein kinase C-iota (PKC-ι) and its specific inhibitor, 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S), in regulating beta-catenin stability and suppressing its signaling in liver cancer cells. We assessed the pathways affected by PKC-ι inhibition by cell proliferation assays, Western blotting, Co-immunoprecipitation, and siRNA knockdowns. HepG2 liver cancer cells were seeded 8×10⁴ per well and treated with ICA-1S (10-50 µM) for 72 hours. Untreated wells served as controls. In comparison, we achieved PKC-ι gene knockdown with 80 nM siRNA using polymer-based transfection reagent siTran 2.0. We then assessed protein expression by Western blotting and normalized it to beta-actin. The dose-response curve of ICA-1S showed a 15%, 35%, 53%, 55%, and 63% reduction in proliferation at concentrations of 10 µM, 20 µM, 30 µM, 40 µM, and 50 µM, respectively, after three days of treatment. Moreover, treatment with 20 µM ICA-1S increased cytochrome C (39%), Bcl-2 Interacting Mediator of cell death (BIM) (25%), cleaved Caspase-3 (24%), and cleaved Poly (ADP-ribose) polymerase (PARP) (24%), while reducing total PARP (35%), indicating an intrinsic apoptotic response. ICA-1S reduced beta-catenin stability, suggesting that PKC-ι maintains the anti-beta-catenin degradation complex. We also observed the downstream effectors of the WNT/beta-catenin signaling pathway in response to 20 µM ICA-1S, which resulted in 53% upregulation of Axin-1, 37% downregulation of disheveled segment polarity protein-3 (DVL3), and 55% and 63% downregulation of Low-density lipoprotein receptor-related protein-6 (LRP6) and phospho-LRP6 levels, respectively. Epithelial cadherin and tumor protein p53 were upregulated by 41% and 13%. We have shown in this study that ICA-1S could inhibit proliferation and induce apoptosis in liver cancer cells by destabilizing beta-catenin and disrupting the WNT/beta-catenin signaling pathway. Our findings identify PKC-ι as a promising therapeutic target, supporting the further development of ICA-1S or related compounds as potential anti-HCC agents that can overcome pathway-specificity challenges in liver cancer therapy.