Abstract 5216: Atypical PKC inhibitors ICA-1S and ζ-Stat show inhibition of neuroblastoma cell proliferation

Abstract

Abstract Neuroblastoma, the pediatric cancer of the early nerve cells, has the tendency to become resistant to traditional therapeutics due to its highly heterogeneous nature. Atypical Protein Kinase C-iota (PKC- ι) has been found to promote cell proliferation in neuroblastoma cells through the PKC- ι /Cdk7/Cdk2 signaling pathway. In this study we utilized various cell lines; I-type (intermediate) CHP-212& BE(2)-C, N-types (neuroblastic); BE(2)-M17& SH-SY5Y and S-types (substrate adherent); SK-N-BE(2) & LAI-5s, representing neuroblastoma heterogeneity to investigate the use of aPKC inhibitors as potential neuroblastoma therapeutics. The aPKC inhibitors, 5-amino-1-2,3-dihydroxy-4-(methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) (PKC- ι specific) and 8-hydroxy-1,3,6-naphthalenetrisulfonic acid (ζ-Stat) (PKC- ζ specific), both display the ability to reduce neuroblastoma proliferation in a cell type specific manner. ICA-1S was shown to reduce proliferation of BE(2)-C, BE(2)-M17, LAI-5s and SK-N-BE(2) to varying degrees. ζ-Stat was able to consistently reduce proliferation of BE(2)-C, BE(2)-M17, SK-N-BE(2), LAI-5s. The degree of proliferation inhibition varied by drug and cell line. Increasing concentration of ICA-1S displayed an inverse relationship between increasing concentration and a decrease in cell proliferation in the four cell lines previously mentioned. ζ-Stat showed a similar relationship to ICA-1S, however had a lower level inhibition than ICA-1S. WST-1 analysis showed that both drugs displayed varying degrees of toxicity in a cell line dependent manner. The results show that I-type cell lines showed the most linearly proportional toxicity to ICA-1S and ζ-Stat in that increasing concentration increased drug toxicity. N-type cell lines displayed a disproportional relationship between concentration and toxicity. The S-type cell lines followed suit with the N-type in their varying degrees of toxicity. Western blot analysis shows that both inhibitors were effective in reducing the expression of total aPKC levels. We observed the downregulation of various mesenchymal markers using the inhibitors. In particular, ICA-1S appears to have a high percentage of decreased expression of N-cadherin. We also accessed the degree of phosphorylation of Vimentin, S33, S39 and S56 positions which are crucial for its activation. In summary both inhibitors prove to be promising potential neuroblastoma therapeutics. The drugs appear to behave in a more cytostatic manner inhibiting the proliferation of neuroblastoma cells. Further western blots ae still ongoing to determine other pathways through which the drugs act to decrease proliferation. Ongoing experiments include the analysis of tumors collected from mouse models to ascertain the effects of the drugs in vivo as well as the analysis of human tumor samples to ascertain the expression of aPKCs and other markers in patient populations. Experiments also include the use of gene expression knockdowns to compare with the effects from the inhibitors as well as the use of microscopy techniques; fluorescent and electron miscopy to observe the localization of mesenchymal and proliferative markers. Citation Format: Sloan Breedy, Wishrawana S. Ratnayake, Christopher Apostolatos, Avijit Dey, Mildred Acevedo-Duncan. Atypical PKC inhibitors ICA-1S and ζ-Stat show inhibition of neuroblastoma cell proliferation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5216.