TP53 R249S mutation in hepatic organoids captures the predisposing cancer risk

Abstract

Background and Aims: Major genomic drivers of hepatocellular carcinoma (HCC) are nowadays well recognized, although models to establish their roles in human HCC initiation remain scarce. Here, we used human liver organoids in experimental systems to mimic the early stages of human liver carcinogenesis from the genetic lesions of TP53 loss and L3 loop R249S mutation. In addition, chromatin immunoprecipitation sequencing (ChIP‐seq) of HCC cell lines shed important functional insights into the initiation of HCC consequential to the loss of tumor‐suppressive function from TP53 deficiency and gain‐of‐function activities from mutant p53. Approach and Results: Human liver organoids were generated from surgical nontumor liver tissues. CRISPR knockout of TP53 in liver organoids consistently demonstrated tumor‐like morphological changes, increased in stemness and unrestricted in vitro propagation. To recapitulate TP53 status in human HCC, we overexpressed mutant R249S in TP53 knockout organoids. A spontaneous increase in tumorigenic potentials and bona fide HCC histology in xenotransplantations were observed. ChIP‐seq analysis of HCC cell lines underscored gain‐of‐function properties from L3 loop p53 mutants in chromatin remodeling and overcoming extrinsic stress. More importantly, direct transcriptional activation of PSMF1 by mutant R249S could increase organoid resistance to endoplasmic reticulum stress, which was readily abrogated by PSMF1 knockdown in rescue experiments. In a patient cohort of primary HCC tumors and genome‐edited liver organoids, quantitative polymerase chain reaction corroborated ChIP‐seq findings and verified preferential genes modulated by L3 mutants, especially those enriched by R249S. Conclusions: We showed differential tumorigenic effects from TP53 loss and L3 mutations, which together confer normal hepatocytes with early clonal advantages and prosurvival functions.

Heterogeneity characterization of hepatocellular carcinoma based on the sensitivity to 5-fluorouracil and development of a prognostic regression model

“…This phenomenon promoted glycolytic metabolic intensity and cell proliferation in HCC. It was also confirmed that the frequency of the R249S mutation in TP53 revealed the risk of HCC, and TP53 deletion increased the viability of hepatocellular carcinoma cells and the trend of poor prognosis ( Lam et al, 2022 ). Interestingly, the C3 isoform might be a TTN mutation-driven molecular subtype that exhibited a high mutational profile.…”

Fibrotic Phenotype in CRISPR knockout p53 of Hepatic Organoids within a Pro-Carcinogenic Microenvironment

“…Tumorigenic transformation typically requires additional driver mutations or enforced microenvironmental changes [35]. Since this model was derived from adult human paired HCC tumor and adjacent non-tumoral liver tissues, compared to our iPSC-derived eHEPO model, the cells may exhibit slightly different responses due to differences in stemness repository (adult-fetal stem cells, respectively) and cell composition [74].…”

Micro‐Engineered Organoid‐on‐a‐Chip Based on Mesenchymal Stromal Cells to Predict Immunotherapy Responses of HCC Patients

“…HCC PDO culture was performed as in the previous study. [ 7 , 26 ] Samples were minced and digested with 2 mg mL −1 collagenase II (GIBCO) at 37 °C for 1 h. Then, pellets were filtered through a 70‐µm cell strainer (Falcon), mixed with Matrigel (Corning), and cultured in HCC organoid culture medium: Advanced DMEM/F12 medium (GIBCO) was supplemented with 1% Penicillin/Streptomycin (Biological Industries), 1% L‐Glutamine (STEMCELL), 10 m m HEPES (GIBCO), 1:50 B27 supplement (without Vitamin A, GIBCO), 1:100 N2 supplement (GIBCO), 1.25 m m n‐Acetyl‐L‐cysteine (Sigma), 10 m m nicotinamide (Sigma), 50 ng mL −1 recombinant human R‐spondin (Rspo)‐1 (Novoprotein), 50 ng mL −1 recombinant human Epidermal growth factor (EGF) (Novoprotein), 50 ng mL −1 recombinant human Fibroblast growth factor (FGF) 10 (Novoprotein), 50 ng mL −1 recombinant human Hepatocyte growth factor (HGF) (Novoprotein), 10 ng mL −1 Noggin (Novoprotein), 5 µ m A8301 (Abmole) and 10 µ m Y27632 (Abmole). Organoids were digested using collagenase II, washed, and cultured in HCC organoid culture medium with Matrigel for subsequent culture.…”

“…Patient‐derived organoids (PDO) are 3D‐culture models which can proliferate in vitro and retain some key characteristics of the original tumor specimens, [ 7 ] therefore displaying promising potential in application on drug screening and precision therapy. [ 8 ] However, their potential to predict clinical outcomes in patients is still unclear, and the in vitro evaluation model for immunotherapeutic drugs remains lacking.…”