Ubiquitin ligase subunit FBXO9 inhibits V-ATPase assembly and impedes lung cancer metastasis

Liang Liu; Xiaodong Chen; Leilei Wu; Kaizong Huang; Zhenyi Wang; Yaolin Zheng; Cheng Zheng; Zhenshan Zhang; Jiayan Chen; Jiaming Wei; Song Chen; Weilin Jin; Jinfei Chen; Dongping Wei; Yaping Xu

doi:10.1186/s40164-024-00497-4

Ubiquitin ligase subunit FBXO9 inhibits V-ATPase assembly and impedes lung cancer metastasis

Exp Hematol Oncol. 2024 Mar 14;13(1):32. doi: 10.1186/s40164-024-00497-4.

Authors

Liang Liu^{1

2}, Xiaodong Chen³, Leilei Wu¹, Kaizong Huang⁴, Zhenyi Wang⁵, Yaolin Zheng⁶, Cheng Zheng⁵, Zhenshan Zhang⁷, Jiayan Chen⁸, Jiaming Wei⁹, Song Chen^{9

10}, Weilin Jin¹¹, Jinfei Chen¹², Dongping Wei^{13

14}, Yaping Xu¹⁵

Affiliations

¹ Department of Radiation Oncology, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, 200433, China.
² Institute of Clinical Medicine, Zhongshan Hospital, Fudan University, Shanghai, 200032, China.
³ Department of General Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325015, Zhejiang Province, China.
⁴ Department of Clinical Pharmacology Lab, Nanjing First Hospital, Nanjing Medical University, Nanjing, 210006, China.
⁵ Medical Research Center, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325015, Zhejiang Province, China.
⁶ Department of Oncology, Nanjing First Hospital, Nanjing Medical University, Nanjing, 210006, China.
⁷ Department of Radiation Oncology, Shanghai Proton and Heavy Ion Center, Fudan University Cancer Center, Shanghai, 200032, China.
⁸ Department of Radiation Oncology, Fudan University Shanghai Cancer Center, Shanghai, 200032, China.
⁹ Institute of Medicinal Biotechnology, Jiangsu College of Nursing, Huai'an, 223300, Jiangsu, China.
¹⁰ Translational Research Institute, Henan Provincial People's Hospital, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, 450053, Henan, China.
¹¹ Institute of Cancer Neuroscience, Medical Frontier Innovation Research Center, The First Hospital of Lanzhou University, The First Clinical Medical College of Lanzhou University, Lanzhou, 730000, China.
¹² Department of Oncology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 730000, Zhejiang Province, China.
¹³ Medical Research Center, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325015, Zhejiang Province, China. weidongping2001@hotmail.com.
¹⁴ Department of Oncology, Nanjing First Hospital, Nanjing Medical University, Nanjing, 210006, China. weidongping2001@hotmail.com.
¹⁵ Department of Radiation Oncology, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, 200433, China. xuyaping1207@126.com.

Abstract

Background: The evolutionarily conserved protein FBXO9 acts as a substrate receptor for the SKP1-cullin-1-RBX1 ubiquitin ligase and is implicated in cancer, exhibiting either tumor-suppressive or oncogenic effects depending on the specific tumor type. However, their role in lung cancer metastasis remains unclear.

Methods: Lentiviral vectors carrying miRNA-based shRNA sequences for gene-specific knockdown were generated, and Lenti-CRISPR-Cas9 vectors containing gene-specific sgRNA sequences were designed. Gene overexpression was achieved using doxycycline-inducible lentiviral constructs, while gene knockdown or knockout cells were generated using shRNA and CRISPR-Cas9, respectively. Functional assays included migration, clonogenic survival assays, tumor sphere assays, and protein interaction studies using mass spectrometry, immunoprecipitation, and immunoblot analysis.

Results: This study identified FBXO9 as a crucial regulator that suppresses lung cancer cell migration, tumor sphere growth and restricts metastasis. We showed that FBXO9 facilitates the ubiquitination of the catalytic subunit A (ATP6V1A) of the Vacuolar-type H⁺-ATPase (V-ATPase), resulting in its interaction with the cytoplasmic chaperone HSPA8 and subsequent sequestration within the cytoplasm. This process hinders the assembly of functional V-ATPase, resulting in reduced vesicular acidification. In contrast, depletion of FBXO9 reduced ATP6V1A ubiquitination, resulting in increased V-ATPase assembly and vesicular acidification, thus promoting pro-metastatic Wnt signaling and metastasis of lung cancer cells. Furthermore, we demonstrated the effectiveness of inhibitors targeting V-ATPase in inhibiting lung cancer metastasis in a mouse model. Finally, we established a correlation between lower FBXO9 levels and poorer survival outcomes in patients with lung cancer.

Conclusion: These findings collectively elucidate the critical role of FBXO9 in regulating V-ATPase assembly and provide a molecular basis for FBXO9's function in inhibiting lung cancer metastasis. This highlights the potential therapeutic opportunities of FBXO9 supplementation.

Keywords: Cancer stemness; FBXO9; Metastasis; Migration; Ubiquitination; V-ATPase assembly.

Abstract

Grants and funding