Direct targeting of HSP90 with daurisoline destabilizes β-catenin to suppress lung cancer tumorigenesis

Highlights

• Functional screening identifies daurisoline as a novel anticancer agent in vitro and in vivo. .

• Daurisoline suppresses lung tumorigenesis by inducing cell cycle arrest. .

• Daurisoline destabilizes β-catenin to inhibit c-Myc and cyclin D1 expression by directly targeting HSP90.

Abstract

Lung cancer is the most frequent cancer worldwide with a poor prognosis. Identification of novel cancer targets and useful therapeutic strategies without toxicity are urgently needed. In this study, we screened natural products for anticancer bioactivity in a library consisting of 429 small molecules. We demonstrated for the first time that daurisoline, a constituent of Rhizoma Menispermi, repressed lung cancer cell proliferation by inducing cell cycle arrest at the G1 phase. Furthermore, daurisoline was found not only to suppress the growth of lung tumor xenografts in animals without obvious side effects, but also to inhibit cell migration and invasion. Mechanistically, quantitative proteomics and bioinformatics analyses, Western blotting and qRT-PCR confirmed that daurisoline exerted its anticancer effects by inhibiting the expression levels of β-catenin and its downstream targets c-myc and cyclin D1. Furthermore, our data from Drug Affinity Responsive Target Stability (DARTS), isothermal titration calorimetry (ITC) and a series of functional assays demonstrated that daurisoline could target HSP90 directly and disrupt its interaction with β-catenin, therefore increasing the ubiquitin-mediated proteasomal degradation of β-catenin. This study reveals that daurisoline could be a promising therapeutic strategy for the treatment of lung cancer.

Introduction

Aberrant Wnt/β-catenin signaling is involved in the pathological processes of multiple human diseases including cancer and maybe a potential therapeutic target for cancer treatment [1]. As a key component in Wnt/β-catenin signaling, β-catenin is phosphorylated by casein kinase I (CK1) and the complex consisting of glycogen synthase kinase 3β (GSK3β), adenomatous polyposis coli (APC) and axin in the absence of Wnt, after which it undergoes ubiquitin-mediated proteasomal degradation [2,3]. In the presence of Wnt ligand, Wnt binds to Frizzled receptors and low-density lipoprotein receptor-related protein 5/6 (LRP5/6) on the cell surface to form a trimer, which transmits signals and activates cytoplasmic dishevelled proteins to attenuate the stability of the APC/axin/GSK-3β complex and prevent β-catenin phosphorylation and degradation [4,5]. Stabilized β-catenin transfers to the nucleus to interact with T-cell factor/lymphoid enhancing factor (TCF/LEF), and then activates transcription of downstream target genes including c-myc and cyclin D1, thereby leading to cancer development and progression [6,7]. Emerging evidence suggests that the stability and transcriptional activity of β-catenin can be affected by a large number of partners that bind to β-catenin and control its activity [8,9]. Therefore, the identification of novel therapeutic strategies that target β-catenin is urgently needed.

Lung cancer is the leading cause of cancer death in the world. Although recently there have been further advances in the understanding of the mechanism of lung tumorigenesis and in the development of anticancer drugs, the treatment outcomes in clinic are still unsatisfactory due to limited efficacy and significant side effects. In this study, we aim to screen for novel anticancer agents with good treatment efficacy and safety in a small molecule library consisting of 429 natural products. Daurisoline, a bis-benzylisoquinoline alkaloid, isolated from the Chinese herbal medicine Rhizoma Menispermi [10], was found to exert the strongest inhibitory effect on lung cancer proliferation. Daurisoline has displayed the potential for treating some diseases, such as focal ischemia-reperfusion injury, arrhythmia and platelet aggregation [11,12], but up until now, the bioactivity of daurisoline in the treatment of cancer and its underlying mechanism are unknown.

Quantitative proteomics coupled with bioinformatic analysis is an excellent strategy for exploring the molecular mechanism of biological processes [13]. With the recent rapid advances in proteomics and chemical biology, Drug Affinity Responsive Target Stability (DARTS), a straightforward method, could be used to identify the target proteins that small molecules directly bind to, without the use of any probe [14,15]. It relies on the principle that the target protein in the cell lysate is resistant to protease digestion when it binds to the ligand molecule, after which mass spectrometry can be used to identify the specific proteins [16]. In the present study, the data from quantitative proteomics and DARTS suggested that daurisoline exerted its anticancer effect through the regulation of β-catenin signaling and may directly target heat shock protein 90 (HSP90), which has been reported to regulate the degradation and expression of β-catenin as an interaction partner [17]. By performing quantitative proteomics, co-immunoprecipitation, and a series of functional assays in vitro and in vivo, we investigated whether daurisoline inhibits the β-catenin pathway and transcription of its downstream target genes to suppress lung tumorigenesis by directly targeting HSP90.