Distinct metabolic phenotype renders β-catenin mutant hepatocellular carcinoma susceptible to treatment-induced ischemia.

Background & Aims: Transarterial chemoembolization (TACE) is the most common treatment for hepatocellular carcinoma (HCC) worldwide; however, response rates and durability vary widely. With the growing armamentarium of therapies for HCC patients, identifying predictors of response to TACE has become increasingly important for a patient population with limited hepatic reserve. We hypothesized that a distinct metabolic phenotype associated with β-catenin pathway mutations render HCC tumors more susceptible to TACE-induced ischemia.

Material And Methods: HCC patients referred for TACE were enrolled in a prospective cohort study at two academic medical centers from April 2016 to October 2021. Liver biopsies were acquired at the time of TACE, and mutational profiles were determined using next generation sequencing. Tumor response was determined by MRI using modified Response Evaluation Criteria in Solid Tumors. HCC cell lines with and without B-catenin mutations were grown in standard and ischemic cell culture conditions (1% O , low nutrient media). Cell viability was measured by WST-1 reagent and Annexin-V PI assay. ATP concentration and metabolites were measured using CellTiter Glo and a YSI biochemical analyzer, respectively. Mitochondrial function was assessed through Seahorse XF Mito Stress Test.

Results: 53 HCC tumors from 50 HCC patients were biopsied prior to TACE, including 22/53 (41.5%) tumors with β-catenin pathway mutations. Despite larger tumor size (4.9 cm vs 3.0 cm p=0.01), tumors with these mutations demonstrated increased rates of complete response after TACE at first imaging (9/22, 40.9% vs 6/31, 19.4%, p=0.06) and best response (12/22, 54.5% vs 7/31, 22.6%, p=0.02), as well as a longer time to tumor progression (median not yet reached vs 8.3 months, p=0.02). modeling confirmed that β-catenin mutant HCC cells have reduced viability (21.4% vs 59.9%, p<0.01) and ATP levels (8.47 vs 4.26 pM/cell, p<0.001) under ischemic conditions compared to β-catenin wild type HCC cells. β-catenin mutant HCC cells had a dramatic increase in their susceptibility to glycolysis inhibition that was not seen in wild type HCC cells (0.09 vs 0.79 IC50 ration for ischemic vs standard conditions, p=0.004), suggesting a change from predominantly aerobic to anaerobic metabolism under ischemia specific to β-catenin mutant. This was further supported by increased sensitivity of β-catenin mutant cells to inhibition of the electron transport chain (43.9% vs 59.5%, p=0.02,) as well as significantly higher basal oxygen consumption rates (0.74 vs 0.39 pmoles/min, p=0.04), maximal respiratory capacity (1.46 vs 0.51 pmoles/min, p=0.01) and ATP-linked respiration (0.58 vs 0.29 pmoles/min, p=0.04).

Conclusions: HCC tumors with activating B-catenin pathway mutations demonstrate a superior response to TACE, driven by enhanced susceptibility to ischemia due to a greater dependence on oxidative phosphorylation for bioenergetic homeostasis. These findings hold the potential to provide a molecular basis for treatment selection in patients with HCC.

Impact And Implications: With the growing armamentarium of locoregional and systemic therapies for patients with HCC, identifying predictors of response to individual therapies has become increasingly important for a patient population with limited hepatic reserve. Current treatment guidelines fail to incorporate molecular biomarkers to inform therapy. In a prospective clinical study of HCC patients undergoing transarterial chemoembolization (TACE), we demonstrated that tumors with activating mutations in the Wnt/B-catenin pathway have increased rates of complete response and longer time to local progression. We further characterized this finding by modeling the post-TACE ischemic environment and demonstrated that B-catenin mutant HCC cells have a distinct metabolic phenotype that renders this subtype more susceptible to ischemia. These findings provide the rationale for genotype-based strategies to enable precision medicine for patients with HCC patients.