The RNA-binding protein La is overexpressed in a number of tumor tissues and is proposed to support tumorigenesis via binding to mRNAs encoding tumor-promoting and anti-apoptotic factors to facilitate their translation. Our data suggest that those mRNAs might need La to be translated mainly in a cap-independent mechanism in cancer cells. Hence, small molecules able to block the binding of La to specific RNAs could represent a novel therapeutic strategy for cancer treatment.
In an attempt toward this approach we developed a high-throughput fluorescence polarization assay to screen small compound libraries for molecules inhibiting the binding of La to an RNA element derived from cyclin D1 mRNA. Herein we describe a robust fluorescence polarization assay and the validation of primary hits by electrophoretic mobility shift assays. Using isothermal titration calorimetry we show that a compound binds to La and molecular modeling suggests an interaction between the compound and the RNA recognition motif of La.
We have shown recently that La protects cells against cisplatin treatment by stimulating the protein synthesis of the anti-apoptotic factor Bcl2. Here we show by RNA immunoprecipitation experiments that one small compound specifically impairs the association of La with Bcl2 mRNA, reduces Bcl2 protein expression and sensitizes cells for cisplatin-induced cell death.
Taken together, herein we report the development of a high-throughput fluorescence polarization assay to identify small compounds able to impair the binding of cancer-associated La to RNAs encoding tumor-promoting and anti-apoptotic factors in vitro and cancer cells.
Citation Format: Gunhild Sommer, Alena Fedarovich, Venkatesh Kota, Yuri K. Peterson, Tilman Heise. Discovery of molecular probes inhibiting La:RNA interactions in cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B25.