Evaluation of the Anti‐Migratory Activity of Small‐molecule Gankyrin Inhibitors in A549 and MDA‐MB‐231 Cells

Abstract

IntroductionGankyrin is a 25 kDa oncoprotein that plays a crucial role in cellular growth, proliferation, and migration. It is highly conserved and has 7 ankyrin repeats which bind to the S6 ATPase subunit of the 26S proteasome. It has been shown that gankyrin exerts its oncogenic activities by interacting with numerous tumor suppressor proteins (TSPs) such as p53 and retinoblastoma protein (Rb), which in turn promotes their proteasomal degradation. While Rb plays an important role in cell adhesion, p53 inhibits Rac/CDC42 which is responsible for lamellipodia and filopodia formation. Gankyrin is also reported to increase the levels of interleukin‐6 (IL‐6) which activates the JAK/STAT3 pathway. The phosphorylation of STAT3 to p‐STAT3 and the subsequent nuclear translocation of p‐STAT3 results in transcription of migratory factors. The inhibition of these gankyrin‐based protein‐protein interactions (PPI) by a small molecule inhibitor, should result in an anti‐migratory effect. Cjoc42 is the first known binder of gankyrin, and since its discovery we have developed two derivatives with an enhanced ability to bind gankyrin, – AFM‐2 and JA‐38 In order to determine if next‐generation gankyrin inhibitors could affect cancer cell migration, we evaluated both compounds in gankyrin overexpressing cell lines A549 and MDA‐MB‐231. Their ability to disrupt multiple gankyrin‐based PPIs is hypothesized to be due to their ability to induce a conformational change in gankyrin upon binding. Various assays such as the scratch assay, Boyden chamber assay, western blotting, co‐immunoprecipitation (co‐IP) assay and fluorescence‐based assays were used to evaluate our hypotheses.MethodsWe evaluated the anti‐migratory potential of the molecules by scratch and Boyden chamber assay. The molecules’ ability to disrupt gankyrin’s interactions with p53, Rb and the S6 proteasome was evaluated by co‐IP assay. Using western blotting, we evaluated the changes in the levels of various TSPs and migratory proteins upon treatment with the molecules. To test our hypothesis of induction of conformational change in gankyrin upon treatment with the molecules, we utilized florescence‐based experiments such as intrinsic fluorescence evaluation and thermal shift.DiscussionThe cell‐based scratch and Boyden chamber assay showed significant decreases in cellular migration in both cell lines upon treatment with the molecules. Co‐IP experiments demonstrated that both molecules disrupt various gankyrin‐based PPIs. Disruption of gankyrin’s interactions with the TSPs would prevent their subsequent proteasomal degradation and subsequently affect the levels of downstream proteins such as IL‐6 and Rac/CDC42. Significant reduction in the migratory proteins and a significant increase in the TSPs was confirmed by western blotting. Lastly, using fluorescence‐based experiments, we believe that the binding of the two molecules caused a conformation change in gankyrin, which could be why gankyrin’s multiple PPIs are disrupted.ConclusionGankyrin plays a key role controlling cellular migratory pathways through its various PPIs. By inducing a conformational change in gankyrin, our molecules disrupt these PPI and exhibit significant anti‐migratory activity which could prevent cancer metastasis.