Lung cancer is one of the leading causes of death worldwide, with lung squamous cell carcinoma (LUSC) being a subtype of non-small cell lung cancer (NSCLC), accounting for 20%-30% of all NSCLC cases. Despite significant advancements in lung cancer treatment in recent years, LUSC still lacks effective targeted therapies, and patients have a poor prognosis. Therefore, identifying new therapeutic targets and strategies has become a key focus of current research.

Cholesterol metabolism plays a crucial role in tumorigenesis and progression. Squalene epoxidase (SQLE) is a key enzyme in the cholesterol synthesis pathway, and previous studies have shown that SQLE has a cancer-promoting role in various cancers. However, its specific mechanism in LUSC remains unclear. Additionally, oxidative stress and ferroptosis have become hot topics in cancer research, and whether SQLE influences LUSC progression by regulating oxidative stress and ferroptosis is an urgent question to be addressed.

Source of the Paper

This paper was co-authored by Guo Li, Lu Chen, Hua Bai, Li Zhang, Jie Wang, and Weimin Li, among others, from institutions such as the National Cancer Center, Chinese Academy of Medical Sciences & Peking Union Medical College, and West China Hospital, Sichuan University. The paper was published on May 8, 2024, in the journal Precision Clinical Medicine, with the DOI 10.1093/pcmedi/pbae011.

Research Process

1. Expression and Prognostic Analysis of SQLE in LUSC

The researchers first analyzed lung adenocarcinoma (LUAD) and LUSC samples from the GEO and TCGA databases to investigate the expression level of SQLE and its relationship with patient prognosis. The results showed that SQLE was significantly overexpressed in LUSC tissues, and patients with high SQLE expression had a worse prognosis. Additionally, immunohistochemical analysis further confirmed the high expression of SQLE in LUSC tissues and indicated that SQLE expression levels were positively correlated with tumor stage.

2. The Impact of SQLE on LUSC Cell Proliferation, Migration, and Invasion

To explore the function of SQLE in LUSC cells, the researchers constructed SQLE knockdown (SQLE-KD) and overexpression (SQLE-OE) cell models. Experimental results showed that SQLE-KD significantly inhibited the proliferation, migration, and invasion abilities of LUSC cells, while SQLE-OE promoted these phenotypes. Further wound healing and Transwell assays also confirmed the role of SQLE in promoting LUSC cell migration and invasion.

3. Therapeutic Effects of SQLE Inhibitors on LUSC Cells

The researchers tested the inhibitory effects of two SQLE inhibitors, terbinafine (TBF) and NB-598, on LUSC cells. CCK-8 assays and Transwell assays demonstrated that both inhibitors significantly suppressed the proliferation and invasion abilities of LUSC cells. Moreover, animal experiments showed that TBF treatment significantly reduced the volume and weight of subcutaneous xenograft tumors in mice, further validating the anti-tumor effects of SQLE inhibitors.

4. Mechanisms of Cell Death Induced by SQLE Knockdown

To uncover the mechanisms by which SQLE knockdown leads to LUSC cell death, the researchers performed RNA-seq and GSEA analysis, revealing that SQLE-KD primarily activated the apoptosis pathway. Further flow cytometry and JC-1 assays confirmed that SQLE-KD promoted LUSC cell apoptosis by inducing mitochondrial membrane potential depolarization and reactive oxygen species (ROS) accumulation.

5. The Impact of SQLE Knockdown on Ferroptosis

Although SQLE-KD induced ROS accumulation, the researchers found that SQLE-KD simultaneously upregulated the expression of glutathione peroxidase 4 (GPX4) and the cystine/glutamate antiporter (System Xc-, XCT), thereby inhibiting lipid peroxidation and ferroptosis. This suggests that SQLE-KD activates antioxidant pathways to counteract the toxicity of ROS, maintaining cell survival.

6. Combination Therapy of SQLE Inhibitors and Ferroptosis Inducers

To overcome ferroptosis resistance caused by SQLE-KD, the researchers combined the SQLE inhibitor TBF with the ferroptosis inducer RSL3. Experimental results showed that the combination therapy significantly inhibited the proliferation and migration abilities of LUSC cells. Animal experiments further confirmed that the combination of TBF and RSL3 significantly reduced the volume and weight of subcutaneous xenograft tumors in mice, indicating the potential application of combination therapy in LUSC treatment.

Key Findings

1. SQLE is highly expressed in LUSC and associated with poor prognosis: SQLE was significantly overexpressed in LUSC tissues, and patients with high SQLE expression had a worse prognosis.
2. SQLE promotes LUSC cell proliferation, migration, and invasion: SQLE-KD significantly inhibited the proliferation, migration, and invasion abilities of LUSC cells, while SQLE-OE promoted these phenotypes.
3. SQLE inhibitors suppress LUSC cell growth: TBF and NB-598 significantly inhibited the proliferation and invasion abilities of LUSC cells, and TBF treatment significantly reduced the volume and weight of subcutaneous xenograft tumors in mice.
4. SQLE-KD inhibits LUSC cell growth by inducing ROS accumulation and apoptosis: SQLE-KD promoted LUSC cell apoptosis by inducing mitochondrial membrane potential depolarization and ROS accumulation.
5. SQLE-KD activates antioxidant pathways to inhibit ferroptosis: SQLE-KD upregulated the expression of GPX4 and XCT, inhibiting lipid peroxidation and ferroptosis.
6. Combination therapy of SQLE inhibitors and ferroptosis inducers significantly suppresses LUSC growth: The combination of TBF and RSL3 significantly inhibited the proliferation and migration abilities of LUSC cells and reduced the volume and weight of subcutaneous xenograft tumors in mice.

Conclusion

This study reveals the important role of SQLE in LUSC, demonstrating that SQLE influences LUSC progression by regulating oxidative stress and ferroptosis. SQLE inhibitors TBF and NB-598 inhibit LUSC cell growth by inducing ROS accumulation and apoptosis. Additionally, SQLE-KD activates antioxidant pathways to inhibit ferroptosis, while the combination of SQLE inhibitors and the ferroptosis inducer RSL3 significantly enhances anti-tumor effects. These findings provide new targets and strategies for LUSC treatment.

Research Highlights

1. High expression of SQLE in LUSC is associated with poor prognosis: This is the first systematic study to reveal the expression characteristics and clinical significance of SQLE in LUSC.
2. SQLE influences LUSC progression by regulating oxidative stress and ferroptosis: This study provides new insights into the molecular mechanisms of SQLE in LUSC, offering a fresh perspective on the pathophysiology of LUSC.
3. Combination therapy of SQLE inhibitors and ferroptosis inducers: The study proposes a new therapeutic strategy combining SQLE and ferroptosis targeting, providing a novel approach for LUSC treatment.

Additional Valuable Information

The SQLE inhibitors TBF and NB-598 used in this study have shown anti-tumor effects in other types of cancer, providing further evidence for the clinical application of SQLE inhibitors. Additionally, the researchers developed SQLE knockdown and overexpression cell models, offering important experimental tools for future studies on the function and mechanisms of SQLE.