Disruption of Gankyrin‐Mediated Epithelial‐Mesenchymal Transition Due to Small Molecule Binding

Abstract

IntroductionGankyrin is a 25 kDa protein that plays a crucial role in cellular growth, proliferation, metastasis, and epithelial‐mesenchymal transition (EMT). It is a highly conserved protein which has 7 ankyrin repeats that binds to the S6 ATPase subunit of the 26S proteasome. It has been shown that gankyrin exerts its oncogenic activities by interacting with a variety of tumor suppressor proteins (TSPs) such as p53 and retinoblastoma protein (Rb). The interaction between gankyrin and certain TSPs promotes their proteasomal degradation. Specifically, the degradation of Rb appears to play a role in gankyrin‐mediated EMT which also contributes towards the highly metastatic nature of gankyrin‐overexpressing cancers. Recently, a small molecule binder of gankyrin has shown to inhibit gankyrin’s ability to aid in the degradation of certain TSPs. It has been previously suggested that small molecule binding of gankyrin induces a conformational change in gankyrin’s structure, which may disrupt its interactions with various TSPs, including Rb. Therefore, our work seeks to show that a small molecule can induce a conformational change which can ultimately affect gankyrin’s ability to induce EMT in certain cancers. Our work is supported by results from fluorescence experiments, western blotting, EMT assays and thermal shift assays.MethodsIn this research, we hypothesize that the conformational change brought about by the small molecule, not only disrupts gankyrin’s binding with the TSPs, but also with proteins that are involved in gankyrin mediated EMT in certain gankyrin overexpressing cell lines. The activity of the proteins involved in EMT, such as STAT3, is also regulated by certain TSPs such as Rb. To test our hypothesis, we performed a series of cell based assays to determine the dose‐dependent effect of a small molecule on TGF‐β induced EMT in gankyrin overexpressing cell lines. We will also perform a series of western blotting and co‐immunoprecipitation (co‐IP) assays to observe the effects of the small molecule on the levels and activity of specific EMT‐related proteins, such as E‐cadherin. We are also seeking to observe the effects of small molecule binding to gankyrin on its interaction with the proteasome, certain TSPs and specific EMT proteins.DiscussionThe cell based assay showed a dose dependent decrease in the TGF‐β induced EMT in gankyrin overexpressing cell lines, in presence of a small molecule. Fluorescence‐based and thermal shift assays strongly suggest that small molecule binding of gankyrin induces a conformational change. Western blot and co‐IP experiments are currently underway.ConclusionGankyrin plays the role of a key middle link which mediates both cancer cell proliferation and metastasis. It achieves this by interacting with and regulating both TSPs and EMT‐associated proteins.