Targeting squalene epoxidase in breast-to-brain metastasis: A new therapeutic target for brain extravasation and colonization using animal models.
Authors: Lee Pony Yu-Ling , Lin Kun-Yuan , Shen Chien-Chang , Chang Chao Di , Cheng Shan-Yun , Hung Ya Wen , Yang Chih-Chieh , Yeh Jia-Yun , Yu Yung-Lung , Wang Shu-Ping 
Published: June 2023

1084

Background: Brain metastasis is the most common malignancy of the central nervous system which causes severe morbidity and mortality in multiple cancer types of patients and represents an unmet medical need. Several critical steps are required for a successful brain metastasis, including local invasion, intravasation, dissemination, extravasation, and colonization. Extensive research has been conducted to elucidate the mechanism of cancer metastasis with limited information toward how cancer cells extravasate and colonize. Methods: To understand the underlying molecular mechanisms and applications of molecular targets for brain metastasis therapy, murine models are employed for investigation of brain extravasation and colonization. To investigate the roles of SQLE in breast-to-brain metastasis in vivo, we silenced SQLE expression directly with lentiviral shRNA in the brain metastatic MDA-MB-231-BR cell line (231-BR/shSQLE) and used 231-BR cells expressing a scramble shRNA (231-BR/shScr) as the control. Brain metastases were induced by intracardiac, orthotopic, or direct intracranial injections of 231-BR/shSQLE or 231-BR/shScr cells into immune deficient mice. The essential roles of SQLE in the specific step(s) of breast-to-brain metastatic process were evaluated by ex vivo immunofluorescence analysis of brain slices from the animals. To verify SQLE as an oncogenic factor that can be selected as a potential therapeutic target in suppressing breast-to-brain metastasis, we evaluated the inhibitory effects of NB-598 (a SQLE inhibitor) in both the 231-BR orthotopic and intracardiac mouse models. Results: Recently, we identified a novel mechanism by which squalene epoxidase (SQLE), the second rate-limiting enzyme in the cholesterol biosynthesis, plays a critical role in the processes of breast cancer metastasized to the brain, especially in brain extravasation and colonization. Interestingly, the pharmacologic inhibition of SQLE has been widely used against fungal infections, and the next-generation SQLE inhibitors have been recently shown to exert an anticancer effect. Our data demonstrated that SQLE is essential for 231-BR cells to extravasate into the parenchyma as well as the formation of micro and macro-metastases in the brain. Interestingly, we found that astrocytes play a key role in supporting 231-BR-developed brain macro-metastasis. In vitro blood-brain barrier (BBB) models further demonstrated the critical roles of SQLE in promoting 231-BR cell invasion and penetration through BBB. Inhibition of SQLE by NB-598 demonstrated anti-tumor proliferation and anti-metastasis. Conclusions: Pharmacologic inhibition of SQLE by NB-598 suppressed 231-BR tumor growth at the mammary fat pad and distal metastases to organs, suggesting that targeting SQLE represents a therapeutic opportunity for breast cancer metastases.