Abstract 333: Identification of a natural product small molecule inhibitor against Wnt/β-catenin signalling pathway
Authors: Dar Mohd Saleem , Hussain Aehtesham , Singh Paramjeet , Hassan Qazi Parvaiz , Monga Satdarshan Paul , Dar* Mohd Jamal 
Published: July 2017

Wnt/β-catenin signalling pathway plays an important role in many processes like cell proliferation, differentiation, regeneration, carcinogenesis and regulates stem cell pluripotency. β-Catenin, the central component of Wnt/β-catenin signalling pathway has a vast number of binding partners and thus plays different roles in the cells. Mutations of β-catenin are seen in many cancers, including liver cancer, colorectal cancer, lung carcinoma, ovarian cancer and malignant breast tumours. Till date there is not even a single small molecule in clinical use that can block the β-catenin activity or the activities of this signalling pathway. In this study, we established many assays for screening small molecule inhibitors of this signalling pathway. Initially, we created a library of β-catenin deletion and point mutants and carried out a comprehensive comparative analysis of these mutants by luciferase reporter assays, GFP-fluorescence, immunoblotting and protein-protein interactions. We then screened many known and unknown natural and synthetic molecules against the wnt/β-catenin pathway and got 2 hits: C-18 and 055A, bioactive metabolites from actinomycetes. The IC50 (cell toxicity) of these molecules along with salinomycin, a known ionophore and inhibitor of β-catenin, were determined in different human cancer cells by MTT assay. The IC50 of 055A, C-18 and salinomycin in HepG2 cells was 7.5µM, 9.7µM and 25µM respectively. Interestingly, C-18 was ineffective against non-cancerous cell line (HEK-293 cells) even upto 60µM. This shows that C-18 is a very promising hit because it is active against cancer cells and spares non-cancerous cells. The sub lethal doses of these molecules were then tested for β-catenin mediated transcription by luciferase reporter assay in HepG2 cells which harbours a β-catenin deletion responsible for enhanced β-catenin mediated transcriptional activity. Our results show that C-18 and 055A significantly block the β-catenin activity in a dose dependent manner. The inhibition of wnt/β-catenin pathway by C-18 was observed at lower nanomolar concentrations in comparison to 055A and salinomycin which shows activity in micromolar concentrations. The β-catenin target gene expression analysis showed marked decrease in cyclinD1 by immunoblot analysis upon treatment of HepG2 cells with C-18. Interestingly, C-18 treated HepG2 cells failed to show any apoptosis as confirmed by Parp-1 and caspase-3 immunoblotting indicating that C-18 can be targeted to block the activity of wnt/β-catenin pathway, and could act as a lead molecule. Therefore, it warrants further experimentation and validation which are underway to draw the further conclusions.

Citation Format: Mohd Saleem Dar, Aehtesham Hussain, Paramjeet Singh, Qazi Parvaiz Hassan, Satdarshan Paul Monga, Mohd Jamal Dar*. Identification of a natural product small molecule inhibitor against Wnt/β-catenin signalling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 333. doi:10.1158/1538-7445.AM2017-333