RELA Ablation Contributes to Progression of Hepatocellular Carcinoma with TP53R249S Mutation and is a Potential Therapeutic Target

Genome‐wide CRISPR/Cas9 based screening identified RELA as a key tumor suppressor in TP53R249S‐mutant HCC. Its loss promotes tumorigenesis and metastasis via DVL1‐mediated Wnt/β‐catenin activation, while its agonist betulinic acid suppresses tumor progression. Abstract TP53 mutations are highly associated with hepatocellular carcinoma (HCC), a common and deadly cancer. However, few primary drivers in the progression of HCC with mutant TP53 have been identified. To uncover tumor suppressors in human HCC, a genome‐wide CRISPR/Cas9‐based screening of primary human hepatocytes with MYC and TP53R249S overexpression (MT‐PHHs) is performed in xenografts. The screen identified RELA as one of the most significant genes, besides NF2 and CSK, two known tumor suppressor genes (TSG) in HCC. Ablation of RELA increased the expression of genes related to cell cycling and stemness in MT‐PHHs, and induced PHHs to transform into HCC in situ in Fah‐deficient immunodeficient mice. Additionally, loss of RELA facilitated HCC metastasis via Epithelial‐Mesenchymal Transition (EMT). Clinically, low RELA expression is positively associated with poor prognosis and large tumor size in HCC patients. In terms of its underlying mechanism, reduced RELA expression promoted DVL1 expression, thereby enhancing β‐catenin nuclear translocation, and thus strengthening Wnt/β‐catenin signaling. Excitingly, betulinic acid (BetA), a RELA agonist, increased RELA activation and suppressed both growth and metastasis of hepatoma cells with TP53R249S overexpression in xenografts. This study reveals RELA as a tumor suppressor in HCC with TP53R249S overexpression, offering a potential therapeutic target. Genome-wide CRISPR/Cas9 based screening identified RELA as a key tumor suppressor in TP53 R249S -mutant HCC. Its loss promotes tumorigenesis and metastasis via DVL1-mediated Wnt/β-catenin activation, while its agonist betulinic acid suppresses tumor progression. Abstract TP53 mutations are highly associated with hepatocellular carcinoma (HCC), a common and deadly cancer. However, few primary drivers in the progression of HCC with mutant TP53 have been identified. To uncover tumor suppressors in human HCC, a genome-wide CRISPR/Cas9-based screening of primary human hepatocytes with MYC and TP53 R249S overexpression (MT-PHHs) is performed in xenografts. The screen identified RELA as one of the most significant genes, besides NF2 and CSK, two known tumor suppressor genes (TSG) in HCC. Ablation of RELA increased the expression of genes related to cell cycling and stemness in MT-PHHs, and induced PHHs to transform into HCC in situ in Fah-deficient immunodeficient mice. Additionally, loss of RELA facilitated HCC metastasis via Epithelial-Mesenchymal Transition (EMT). Clinically, low RELA expression is positively associated with poor prognosis and large tumor size in HCC patients. In terms of its underlying mechanism, reduced RELA expression promoted DVL1 expression, thereby enhancing β-catenin nuclear translocation, and thus strengthening Wnt/β-catenin signaling. Excitingly, betulinic acid (BetA), a RELA agonist, increased RELA activation and suppressed both growth and metastasis of hepatoma cells with TP53 R249S overexpression in xenografts. This study reveals RELA as a tumor suppressor in HCC with TP53 R249S overexpression, offering a potential therapeutic target. Advanced Science, Volume 12, Issue 48, December 29, 2025.