Silibinin is a suppressor of the metastasis-promoting transcription factor ID3

Abstract

Background: ID3 (inhibitor of DNA binding/differentiation-3) is a transcription factor that enables metastasis by promoting stem cell-like properties in endothelial and tumor cells. The milk thistle flavonolignan silibinin is a phytochemical with anti-metastatic potential through largely unknown mechanisms. Hypothesis/purpose: We have mechanistically investigated the ability of silibinin to inhibit the aberrant activation of ID3 in brain endothelium and non-small cell lung cancer (NSCLC) models. Methods: Bioinformatic analyses were performed to investigate the co-expression correlation between ID3 and bone morphogenic protein (BMP) ligands/BMP receptors (BMPRs) genes in NSCLC patient datasets. ID3 expression was assessed by immunoblotting and qRT-PCR. Luciferase reporter assays were used to evaluate the gene sequences targeted by silibinin to regulate ID3 transcription. In silico computational modeling and LanthaScreen TR-FRET kinase assays were used to characterize and validate the BMPR inhibitory activity of silibinin. Tumor tissues from NSCLC xenograft models treated with oral silibinin were used to evaluate the in vivo anti-ID3 effects of silibinin. Results: Analysis of lung cancer patient datasets revealed a top-ranked positive association of ID3 with the BMP9 endothelial receptor ACVRL1/ALK1 and the BMP ligand BMP6. Silibinin treatment blocked the BMP9-induced activation of the ALK1-phospho-SMAD1/5-ID3 axis in brain endothelial cells. Constitutive, acquired, and adaptive expression of ID3 in NSCLC cells were all significantly downregulated in response to silibinin. Silibinin blocked ID3 transcription via BMP responsive elements in ID3 gene enhancers. Silibinin inhibited the kinase activities of BMPRs in the micromolar range, with the lower IC50 values occurring against ACVRL1/ALK1 and BMPR2. In an in vivo NSCLC xenograft model, tumoral overexpression of ID3 was completely suppressed by systematically achievable oral doses of silibinin. Conclusions: ID3 is a largely undruggable metastasis-promoting transcription factor. Silibinin is a novel suppressor of ID3 that may be explored as a novel therapeutic approach to interfere with the metastatic dissemination capacity of NSCLC

The work in the Menendez laboratory is supported by the Ministerio de Ciencia e Innovación (MCIN, grants PID2019-104055GB-I00 and PID2022-141955OB-I00 to Javier A. Menendez, Plan Nacional de I + D + i, funded by the European Regional Development Fund, Spain) and the Emerging Research Group SGR 2021 01,507 to Begoña Martin-Castillo from the Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR, Generalitat de Catalunya). Elisabet Cuyàs holds a “Miguel Servet” research contract (CP20/00,003) from the Instituto de Salud Carlos III (Spain) and is supported by the Grant PI22/00,297 (Instituto de Salud Carlos III, Proyectos de I + D + I en Salud, Acción Estratégica en Salud 2021–2023, founded by the European Regional Development Fund, Spain). The work in the Jose A. Encinar laboratory is supported by the Spanish Ministry of Economy and Competitiveness (MINECO, Grant RTI2019–096,724-B-C21 ) and the Generalitat Valenciana ( PROMETEO/2021/059 ). Eila Serrano-Hervás holds an INVESTIGO research contract (2022 INV-1 00,001, Next Generation Catalunya, Next Generation EU) from the Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR, Generalitat de Catalunya). Eduard Teixidor holds a “Rio Hortega” research contract (CM22/00,276, Proyectos de I + D + I en Salud, Acción Estratégica en Salud 2021–2023, founded by the European Regional Development Fund, Spain) from the Instituto de Salud Carlos III (Spain)