Nitazoxanide inhibits acetylated KLF5-induced bone metastasis by modulating KLF5 function in prostate cancer
Qingqing Huang1, Mingcheng Liu1, Duo Zhang1
1Department of Human Cell Biology and Genetics, School of Medicine, Southern University of Science and Technology, 1088 Xueyuan Blvd, Shenzhen, 518055, China.
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 KLF5K369Q, which mimics Ac-KLF5, to screen 1987 FDA-approved drugs for invasion suppression. Luciferase- and KLF5K369Q-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 KLF5K369Q-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 KLF5K369Q. NTZ attenuated the function of KLF5K369Q 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, KLF5K369Q bound to the promoter of MYBL2 to activate its transcription, and NTZ attenuated the binding of KLF5K369Q 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.
Insights
Nitazoxanide (NTZ) effectively inhibits prostate cancer bone metastasis by targeting the TGF-β/acetylated KLF5 pathway. This anthelmintic agent shows promise as a therapeutic for bone metastasis and potentially other cancers.
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- Castration-resistant prostate cancer frequently metastasizes to bone, developing therapeutic resistance.
- Transforming growth factor-beta (TGF-β) is crucial in bone metastasis, but direct targeting is challenging.
- TGF-β induces KLF5 acetylation (Ac-KLF5), a key regulator of prostate cancer bone metastasis.
Purpose of the Study:
- To identify inhibitors of KLF5-mediated invasion and bone metastasis.
- To investigate the therapeutic potential of nitazoxanide (NTZ) in prostate cancer bone metastasis.
Main Methods:
- Screened FDA-approved drugs using a KLF5K369Q prostate cancer cell invasion assay.
- Modeled bone metastasis in mice using KLF5K369Q-expressing cells and evaluated using bioluminescence and micro-CT.
- Utilized RNA-sequencing, bioinformatics, and biochemical analyses to elucidate NTZ's mechanism of action.
Main Results:
- Identified nitazoxanide (NTZ) as a potent inhibitor of prostate cancer cell invasion and bone metastasis.
- NTZ inhibited osteoclast differentiation and modulated the expression of 231 genes regulated by KLF5K369Q.
- NTZ directly bound to KLF5 and prevented KLF5K369Q binding to the MYBL2 promoter, inhibiting MYBL2 upregulation.
Conclusions:
- Nitazoxanide (NTZ) is a promising therapeutic agent for prostate cancer bone metastasis driven by the TGF-β/Ac-KLF5 pathway.
- NTZ's mechanism involves inhibiting KLF5-mediated gene regulation and osteoclast differentiation.
- NTZ may be effective for treating bone metastasis in other cancers as well.
