Nitazoxanide inhibits acetylated KLF5-induced bone metastasis by modulating KLF5 function in prostate cancer

Abstract

Background: Castration-resistant prostate cancer often metastasizes to the bone, and such bone metastases eventually become resistant to available therapies, leading to the death of patients. Enriched in the bone, TGF-β plays a pivotal role in bone metastasis development. However, directly targeting TGF-β or its receptors has been challenging for the treatment of bone metastasis. We previously found that TGF-β induces and then depends on the acetylation of transcription factor KLF5 at K369 to regulate multiple biological processes, including the induction of EMT, cellular invasiveness, and bone metastasis. Acetylated KLF5 (Ac-KLF5) and its downstream effectors are thus potential therapeutic targets for treating TGF-β-induced bone metastasis in prostate cancer.

Methods: A spheroid invasion assay was applied to prostate cancer cells expressing KLF5^K369Q, which mimics Ac-KLF5, to screen 1987 FDA-approved drugs for invasion suppression. Luciferase- and KLF5^K369Q-expressing cells were injected into nude mice via the tail artery to model bone metastasis. Bioluminescence imaging, micro-CT), and histological analyses were applied to monitor and evaluate bone metastases. RNA-sequencing, bioinformatic, and biochemical analyses were used to understand nitazoxanide (NTZ)-regulated genes, signaling pathways, and the underlying mechanisms. The binding of NTZ to KLF5 proteins was evaluated using fluorescence titration, high-performance liquid chromatography (HPLC), and circular dichroism (CD) analysis.

Results: NTZ, an anthelmintic agent, was identified as a potent invasion inhibitor in the screening and validation assays. In KLF5^K369Q-induced bone metastasis, NTZ exerted a potent inhibitory effect in preventive and therapeutic modes. NTZ also inhibited osteoclast differentiation, a cellular process responsible for bone metastasis induced by KLF5^K369Q. NTZ attenuated the function of KLF5^K369Q in 127 genes' upregulation and 114 genes' downregulation. Some genes' expression changes were significantly associated with worse overall survival in patients with prostate cancer. One such change was the upregulation of MYBL2, which functionally promotes bone metastasis in prostate cancer. Additional analyses demonstrated that NTZ bound to the KLF5 protein, KLF5^K369Q bound to the promoter of MYBL2 to activate its transcription, and NTZ attenuated the binding of KLF5^K369Q to the MYBL2 promoter.

Conclusions: NTZ is a potential therapeutic agent for bone metastasis induced by the TGF-β/Ac-KLF5 signaling axis in prostate cancer and likely other cancers.

Keywords: Acetylated-KLF5; Bone metastasis; MYBL2; Nitazoxanide; Prostate cancer; TGF-β.