C3G deregulation uncovers a dual role in B-cell lymphoma: tumor suppression and enhanced metastasis via Rap1 and Rac2 signaling.

BACKGROUND

C3G (RapGEF1) is a guanine nucleotide exchange factor that activates Rap1, a small GTPase implicated in hematologic malignancies. We previously showed that C3G GEF activity is self-repressed via its AIR (autoinhibitory region). A lymphoma-associated missense mutation (Y554H) disrupts this inhibition, resulting in constitutive activation. This study aims to investigate the consequences of C3G dysregulation in B-cell lymphoma.

METHODS

Murine C3G mutation Y564H (equivalent to human Y554H) was introduced into the A20 B-cell lymphoma line using CRISPR/Cas9. Rap1 activation, proliferation, apoptosis, ERK1/2 phosphorylation, Rac2 activity, adhesion, migration, invasion, tumorigenicity, and transcriptomic changes were assessed through biochemical assays, in vitro functional studies, in vivo mouse models, and RNA-seq analysis.

RESULTS

A20-C3G-Y564H cells exhibited increased Rap1 activation under both basal and stimulated conditions. Hyperactivation of the C3G-Rap1 pathway impaired proliferation, promoted apoptosis, and was associated with reduced ERK1/2 phosphorylation. Furthermore, Rac2 activity was diminished, correlating with altered adhesion properties. Consistently, cell migration and invasion were enhanced, in correspondence with an increased number of metastatic foci in the liver following tail vein injection into syngeneic BALB/c mice. Notably, reduced C3G expression further augmented the metastatic potential of A20 cells. RNA-seq analysis revealed widespread transcriptional changes involving Rac2 signaling, adhesion, and metastatic pathways.

CONCLUSIONS

C3G plays a dual role in B-cell lymphoma: it acts as a tumor suppressor by inhibiting growth and promoting apoptosis, but may also facilitate metastasis via enhanced motility. This dual effect likely reflects a functional balance between Rap1 and Rac2 signaling. These findings underscore the complexity of C3G-regulated pathways in B cells and suggest that C3G may serve as a potential novel marker in hematologic malignancies.