Abstract 150: Prevalent PKC-ι/ζ signaling is crucial for apoptosis inhibition and invasiveness of glioblastoma through upregulation of Pdk1/Akt1/14-3-3 cascade and Smad2/3
Authors: Ratnayake Wishrawana Sarathi , Khalid Khandker M. , Breedy Sloan , Lajmi Luke , Smalley Tracess , Apostolatos Christopher A. , Hill Robert , Fackrell James , Acevedo-Duncan Mildred 
Published: June 2022

Glioblastoma (GB) is an aggressive form of cancer derived from astrocytes and often found in brain or spinal cord. Poor prognosis of GB has limitations in current therapeutics mainly due to the development of resistance. Our results demonstrated that atypical protein kinase C-iota and zeta (aPKC-ι/ζ) levels were over-expressed in GB cells (U-87 and T-98) and tissue samples compared to normal brain cells/tissues which is correlated with increased invasiveness. The present study shows the downstream effects of siRNA knockdown of PKC-ι/ζ on the expression of aPKCs, E-cadherin, Vimentin, p-Vimentin, 14-3-3 and p-14-3-3, BAD, p-BAD, Pdk1, Akt1, c-Raf, Tak1, Smad2/3 and p-Smad2/3. Downregulation of Vimentin, p-Vimentin (S33 and S56) and increased E-cadherin indicated the downregulation of epithelial-mesenchymal transition (EMT). Proteolytic E-cadherin fragment of 80 kDa indicates the reduction of cancer progression. Result also indicated that SNAIL1, SLUG and PRRX1 transcriptional activities were downregulated as a result of aPKC diminution. These transcription factors are known to induce and promote EMT. 14-3-3 is known to bind Pdk1, BAD, Akt1 and c-Raf and perform various anti-apoptotic tasks. Our results indicated that total 14-3-3 levels were significantly increased as a result of aPKC attenuation along with dephosphorylated BAD. In addition, p-14-3-3, p-BAD, pAkt1, Pdk1 levels were significantly decreased. Unphosphorylated 14-3-3 does not bind with their partners: phosphorylation on conserved S259 and S621 through Tak1 which is essential for its activation. Our results indicated that Tak1 levels significantly decreased as a result of aPKC diminution. In-vitro migration/invasion assays, immunoprecipitation (IP), immunofluorescence and immuno-gold transmission electron microscopic techniques, real time qPCR and Western blot techniques are being used in this study to further analyze role of aPKCs in GB malignancy. Our IP and kinase activity assay data confirmed that PKC-ι heavily interacts with Pdk1 and PKC-ζ interacts with Akt1, thereby activating those two kinases via phosphorylation. Data confirmed that aPKC attenuation triggers multiple pro apoptotic signaling cascades through downregulation of Akt1, Pdk1, 14-3-3 and Smad2/3.We have used a novel PKC-ι specific inhibitor 5-amino-1-(2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) and a PKC-ζ specific inhibitor 8-hydroxy-1,3,6-naphthalenetrisulfonic acid (ζ-Stat) to conduct in-vivo experiments on murine models. Excised tumors will be analyzed for pathways observed in in-vitro experiments. Overall results suggest that aPKCs are crucial for increased invasiveness in GB. Results suggested that aPKC can be targeted to develop customized, tailored therapies for Glioblastoma which merit further research.

Citation Format: Wishrawana Sarathi Ratnayake, Khandker M. Khalid, Sloan Breedy, Luke Lajmi, Tracess Smalley, Christopher A. Apostolatos, Robert Hill, James Fackrell, Mildred Acevedo-Duncan. Prevalent PKC-ι/ζ signaling is crucial for apoptosis inhibition and invasiveness of glioblastoma through upregulation of Pdk1/Akt1/14-3-3 cascade and Smad2/3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 150.