Abstract B048: Activation of PKC inhibits oncogenic KRAS-driven malignancies

The extremely high mutation rate of KRAS in three of the top four deadly cancers (lung, colon, and pancreas) makes it a major target in anticancer drug discovery. However, no effective anti-KRAS inhibitors have yet been developed into clinically useful drugs. Therefore, identifying “druggable” signaling components crucial for KRAS can provide a new approach for targeting KRAS-driven malignancies. We have reported that oncogenic KRAS uniquely elicits a tumorigenic phenotype through downregulation of noncanonical Fzd8/Wnt/Ca2+ signaling. Binding of calmodulin (CaM) to KRAS, but not to H- or N-RAS, appears to be responsible for this major difference. The total intracellular concentration of CaM has been reported to be significantly below the total concentration of its targets, making CaM a limiting factor in their regulation. Therefore, the unique binding of KRAS to CaM, which competes for a limiting pool of CaM, can be sufficient to affect CaM-dependent signaling pathways. Serine-181 in the hypervariable region of KRAS, as the essential domain for KRAS to bind to CaM, has been identified as the phosphorylation site of PKC. The KRAS-V12-S181D (pseudo-phosphorylated) variant lost the ability to suppress CaMKii activity and Fzd8 expression, suggesting that the interaction between KRAS and CaM is an important pathway for KRAS to inhibit Fzd8-mediated noncanonical Wnt/Ca2+ signaling. Thus, we hypothesize to target KRAS selectively by blocking this specific interaction between KRAS and CaM through the activation of PKC. The activation of PKC by a non-tumor promoting PKC activator, prostratin, successfully interrupts the KRAS-CaM interaction, rewires the sequential downstream signaling pathways, and, most importantly, represses the tumor initiation and growth in KRAS-driven cancers. The pancreatic cancer cells treated with Gο6984 (targeting conventional and novel PKCs), but not Gο6976 (targeting conventional PKCs), lost the response to prostratin treatment on activation of CaMKii. Next, each individual of PKC isozyme in conventional and novel subfamilies has been knocked down by siRNA, followed by the treatment of prostratin. The cells, where PKCδ has been knocked down, failed to activate CaMKii in the response to prostratin, when compared to the cells with the depletions of other PKC isoforms. The pancreatic cancer cells where PKCδ, but not PKCα, has been knocked down by shRNA lost their sensitivity to prostratin on cell viability and growth in vitro. The treatment of prostratin increased the phosphorylated PKCδ at serine-299 in KRAS transformed cells, suggesting prostratin acts as a PKCδ activator. The cellular thermal shift assay suggested that, when compared to PMA, prostratin preferentially stabilized PKCδ in a dose-dependent manner. To further define the specificity of prostratin-induced PKC activation on phosphorylation of KRAS, we treated the LSL-KRAS-G12D mice carrying the additional mutation at serine-181 (S181S to A, resistant to phosphorylation) with prostratin post-adenoCre infection. The lung tumors from LSL-KRAS-G12D-S181S mice, but not -S181A ones, showed increased phosphorylated CaMKii in the response to prostratin treatment. The results suggest that activation of certain specific PKC isozymes may provide a novel approach to rewire KRAS-mediated signaling pathway and to sequentially suppress its malignant features in human cancers. Citation Format: Man-Tzu Wang, Jacqueline Galeas, Frank McCormick. Activation of PKC inhibits oncogenic KRAS-driven malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B048.