Natural product (L)-gossypol inhibits colon cancer cell growth by targeting RNA-binding protein Musashi-1

Musashi-1 (MSI1) is an RNA-binding protein that acts as a translation activator or repressor of target mRNAs. The best-characterized MSI1 target is Numb mRNA, whose encoded protein negatively regulates Notch signaling. Additional MSI1 targets include the mRNAs for the tumor suppressor protein APC that regulates Wnt signaling and the cyclin-dependent kinase inhibitor P21WAF−1. We hypothesized that increased expression of NUMB, P21 and APC, through inhibition of MSI1 RNA-binding activity might be an effective way to simultaneously downregulate Wnt and Notch signaling, thus blocking the growth of a broad range of cancer cells. We used a fluorescence polarization assay to screen for small molecules that disrupt the binding of MSI1 to its consensus RNA binding site. One of the top hits was (−)-gossypol (Ki = 476 ± 273 nM), a natural product from cottonseed, known to have potent anti-tumor activity and which has recently completed Phase IIb clinical trials for prostate cancer. Surface plasmon resonance and nuclear magnetic resonance studies demonstrate a direct interaction of (−)-gossypol with the RNA binding pocket of MSI1. We further showed that (−)-gossypol reduces Notch/Wnt signaling in several colon cancer cell lines having high levels of MSI1, with reduced SURVIVIN expression and increased apoptosis/autophagy. Finally, we showed that orally administered (−)-gossypol inhibits colon cancer growth in a mouse xenograft model. Our study identifies (−)-gossypol as a potential small molecule inhibitor of MSI1-RNA interaction, and suggests that inhibition of MSI1's RNA binding activity may be an effective anti-cancer strategy

Identification and Validation of an Aspergillus nidulans Secondary Metabolite Derivative as an Inhibitor of the Musashi-RNA Interaction

This work is licensed under a Creative Commons Attribution 4.0 International License.RNA-binding protein Musashi-1 (MSI1) is a key regulator of several stem cell populations. MSI1 is involved in tumor proliferation and maintenance, and it regulates target mRNAs at the translational level. The known mRNA targets of MSI1 include Numb, APC, and P21WAF-1, key regulators of Notch/Wnt signaling and cell cycle progression, respectively. In this study, we aim to identify small molecule inhibitors of MSI1–mRNA interactions, which could block the growth of cancer cells with high levels of MSI1. Using a fluorescence polarization (FP) assay, we screened small molecules from several chemical libraries for those that disrupt the binding of MSI1 to its consensus RNA. One cluster of hit compounds is the derivatives of secondary metabolites from Aspergillus nidulans. One of the top hits, Aza-9, from this cluster was further validated by surface plasmon resonance and nuclear magnetic resonance spectroscopy, which demonstrated that Aza-9 binds directly to MSI1, and the binding is at the RNA binding pocket. We also show that Aza-9 binds to Musashi-2 (MSI2) as well. To test whether Aza-9 has anti-cancer potential, we used liposomes to facilitate Aza-9 cellular uptake. Aza-9-liposome inhibits proliferation, induces apoptosis and autophagy, and down-regulates Notch and Wnt signaling in colon cancer cell lines. In conclusion, we identified a series of potential lead compounds for inhibiting MSI1/2 function, while establishing a framework for identifying small molecule inhibitors of RNA binding proteins using FP-based screening methodology.NIH R01 CA178831NIH CA191785National Institute of General Medical Science (P01GM084077)University of Kansas Bold Aspiration Strategic Initiative AwardNational Cancer Institute Cancer Center Support Grant P30 CA168524Kansas Bioscience Authority Rising Star AwardNIH AI074856NIH COBREIrving S. Johnson Fund of the University of Kansas Endowmen