Deciphering the oncogenic role of Rac family small GTPase 3 in hepatocellular carcinoma through multiomics integration.

Abstract

Background: Hepatocellular carcinoma (HCC) remains a lethal malignancy due to its molecular complexity and chemoresistance. Rac family small GTPase 3 (RAC3), a tumorigenic GTPase understudied in HCC, drives recurrence via E2F transcription factor 1 (E2F1)-mediated transcriptional activation. This study integrates multiomics and clustered regularly interspaced short palindromic repeats (CRISPR) screening to delineate RAC3's roles. RAC3 overexpression correlates with advanced HCC and patient age, while its knockout suppresses proliferation. Mechanistically, RAC3 dysregulates cell-cycle checkpoints through E2F1 binding. Pharmacological RAC3 inhibition disrupts tumor growth and synergizes with chemotherapy to overcome resistance.

Aim: To explore RAC3's expression, clinical links, and HCC mechanisms via multiomics and functional genomics.

Methods: Multiomic integration of The Cancer Genome Atlas (TCGA), Gene Expression Omnibus, and Genotype-Tissue Expression datasets was performed to analyze RAC3 mRNA expression. Immunohistochemistry quantified RAC3 protein in 108 HCC/adjacent tissue pairs. Kaplan-Meier/Cox regression assessed prognostic significance using TCGA data. CRISPR screening validated RAC3's necessity for HCC proliferation. Functional enrichment identified associated pathways; hTFtarget/JASPAR predicted transcription factors, validated via chromatin immunoprecipitation sequencing (ChIP-seq).

Results: RAC3 exhibited significant mRNA and protein overexpression in HCC tissues, which was correlated with advanced tumor stages and reduced overall survival rates (hazard ratio = 1.82, 95%CI: 1.31-2.53). Genetic ablation of RAC3 suppressed HCC cell proliferation across 16 cell lines. Pathway analysis revealed RAC3's predominant involvement in cell-cycle regulation, DNA replication, and nucleocytoplasmic transport. Mechanistic investigations identified E2F1 as a pivotal upstream transcriptional regulator, and ChIP-seq analysis validated its direct binding to the RAC3 promoter region. These findings suggest that RAC3 drives HCC progression through E2F1-mediated cell-cycle dysregulation.

Conclusion: This study identified RAC3 as a key HCC oncogenic driver; its overexpression links to poor prognosis/resistance. Targeting the RAC3/E2F1 axis offers a new therapy, which highlights RAC3 as a biomarker/target.