Identification and characterization of stemness-relevant prognostic biomarkers regulating tumor progression, metastasis and therapy resistance in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) remains¬ the most prevalent form of primary liver cancer worldwide and represents a major worldwide health-care challenge due to high incidence and mortality. Treatment failure of HCC is significantly influenced by cellular diversity, particularly intratumor heterogeneity. Mounting evidence suggests that cancer stem cells (CSCs) are crucial for hierarchical organization within tumors. CSCs display stemness in the tumor microenvironment (TME) to encourage tumor initiation, progression, metastasis, immune evasion and therapy resistance. Therefore, understanding the molecular processes underlying CSC plasticity, maintenance and treatment tolerance in HCC will provide insights of therapeutic strategies targeting intrinsic properties of LCSCs to enhance the current clinical practice of patients. Recent studies suggest that dysregulation of RNA editing by ADAR1 is implicated in transcriptome diversity of HCC with prognostic values. The processes controlling LCSC generation and maintenance that are dependent on ADAR1, however, remain elusive. Thus, our objective is to uncover the molecular mechanisms behind ADAR1-orchestrated HCC stemness using an ex vivo human hepatocyte differentiation model. The whole transcriptome profiling identified ADAR1-responsive aberrant hyper-editing of GLI1 in HCC rather than transcript abundance, was clinically relevant. The roles played by wild-type and edited GLI1 (GLI1wt versus GLI1edit) in LCSC self-renewal and cancer progression were clarified. GLI1edit was found to lead to an A-to-G (R701G) substitution that allowed GLI1 cytoplasmic-to-nuclear translocation and stabilization because of weak affinity to SUFU and β-TrCP respectively, which in turn upregulated pluripotency genes to initiate tumors and gave rise to a more aggressive phenotype. Activated mitophagy in ADAR1-enriched LCSCs was also discovered through integrated analysis of single-cell transcriptome. GLI1edit facilitated a metabolic shift to oxidative phosphorylation through PINK1-Parkin axis to sustain stem-like state of tumor cells, granting exclusive metastatic and sorafenib-resistant capabilities. Sorafenib frequently proves ineffective as a first-line systemic therapy choice for patients with advanced HCC. Immune-checkpoint inhibitor (ICI)-based therapies have recently revolutionized advanced HCC management although clinical evidence showed that prior treatment with sorafenib influences response to anti-PD-1 in second line with unknown reasons. To investigate the mechanisms underlying cross-resistance to therapy within tumors, transcriptomic profiling was performed in various mouse models exhibiting restricted response to sorafenib. The results revealed that FZD4 is a critical stemness-related gene in HCC tumors and predicts poor prognosis. By applying pharmacological and genetic methods, FZD4 was demonstrated to promote tumor progression and TKIs tolerance. Furthermore, the tumor-infiltrating CD8+ T cells were observed to lose their effector function in FZD4-enriched HCC. Mechanistically, cholesterol metabolic reprogramming in tumor cells directed by WNT5A-FZD4-ROR2 axis induced an immune suppressive TME via CD8+ T-cell dysfunction. Suppression of FZD4 rescued the efficacy of either subsequent anti-PD-1 therapy in sorafenib-resistant tumors or sorafenib-anti-PD-1 combination treatment against HCC. Altogether, two stemness-associated regulators (ADAR1 and FZD4) are identified in the highly heterogeneous HCC. The role of ADAR1 as an active regulator of LCSC properties by editing GLI1 RNA is clarified, and FZD4 is prospectively demonstrated to be a crucial factor in tumor progression, potentially expanding the scope of targeted/ICI therapies for better outcomes.