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

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.