Genetically Determined Metabotype Renders β-catenin Mutant Hepatocellular Carcinomas Susceptible to Treatment Induced Ischemia

Monday, March 6, 2023

12:00 PM – 1:00 PM

Location: Phoenix Convention Center, North Ballroom 120 D

Poster Presenter(s)

JC

Jennifer A. Crainic, BA

Research Assistant
University of Pennsylvania

Disclosure information not submitted.

Author/Co-author(s)

KW

Kelley Weinfurtner, MD (she/her/hers)

Transplant Hepatologist
University of Pennsylvania
Isabela S. Gatmaytan, B.A.

Clinical Research Coordinator
Hospital of the University of Pennsylvania
Nicholas Perkons, MD, PhD

Interventional Radiology Resident
Department of Radiology, Duke University
AG

Alexey Gurevich, MD, MS

Interventional Radiology Resident
Division of Interventional Radiology, Department of Radiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA
DA

Daniel Ackerman, PhD

Research Assistant
University of Pennsylvania
Gregory J. Nadolski, MD

Attending Physician
Penn Image-Guided Interventions (PIGI) Lab, Hospital of the University of Pennsylvania, Division of Interventional Radiology
Stephen Hunt, MD, PhD, FSIR (he/him/his)

Assistant Professor of Radiology
Penn Image-Guided Interventions (PIGI) Lab, Hospital of the University of Pennsylvania
TG

Terence P. Gade, MD PhD

Assistant Professor of Radiology
Penn Image-Guided Interventions (PIGI) Lab, Hospital of the University of Pennsylvania

Disclosure(s):

Kelley Weinfurtner, MD: No financial relationships to disclose

Alexey Gurevich, MD, MS: No financial relationships to disclose

Stephen Hunt, MD, PhD, FSIR: Boston Scientific: Consultant (Ongoing), Speaking and Teaching (Ongoing); Guerbet: Research Grant Recipient (Ongoing); Instylla, Inc.: Research Grant Recipient (Ongoing)

Terence P. Gade, MD PhD: Astra Zeneca: Advisory Committee or Review Panel Member (Terminated); Instylla, Inc.: Research Grant Recipient (Ongoing); Trisalus Life Sciences: Advisory Committee or Review Panel Member (Ongoing)

Purpose: 30-40% of hepatocellular carcinoma (HCC) patients have activating mutations in the β-catenin pathway. Despite larger tumor size, tumors with activating β-catenin pathway mutations have higher rates of complete response, and longer local time-to-progression, following transarterial embolization (TACE) as compared to HCCs without these mutations. The purpose of this study was to explore the molecular mechanisms that may contribute to this response.

Materials and Methods: β-catenin mutant (SNU-398, HepG2) and wild type (Huh7, SNU-449) cell lines were obtained from ATCC. Cells were grown in standard (21% O₂, RPMI, 1% dialyzed FBS, 10 mM glucose, 2.05 mM glutamine) or ischemic (1% O₂, RPMI, 1% dialyzed FBS, 1 mM glucose, 0.5 mM glutamine) conditions. Metabolites were analyzed on a YSI analyzer. ATP levels were quantified using the CellTiter-Glo Assay. Protein expression was measured by western blot. Viability was assessed using annexin/PI flow cytometry. Lactate Dehydrogenase (LDH), β-catenin, and fatty-acid oxidation were inhibited with NCATS-SM1441, XAV939 (Selleckem), and Etomoxir (Selleckem) respectively

Results: HCC cells incubated in standard conditions demonstrated similar viability regardless of genetic background, but the viability of the β-catenin mutant cell lines was significantly lower under ischemia (80.8% to 64.8% , p=0.0037). This difference was associated with marked reductions in ATP levels observed only in the mutant cell lines (8.65 vs 4.44, p=0.0003) and increased bioenergetic stress as evidence by activation of autophagy and sestrin-2. Pharmacologic inhibition of β-catenin restored viability (Mut: 100% vs 115%, p=0.016) and ATP levels (% change in ATP WT vs Mut, -0.80% vs 9.26%, p< 0.0001) in the mutants and also decreased levels of bioenergetic stress. No differences in glycolysis, glutamine metabolism, or fatty acid oxidation were identified between the groups. However, β-catenin mutant cell lines were more reliant on oxidative phosphorylation as seen by a substantial decrease in ATP following electron chain transport inhibition (72.37% vs 54.63%, p=0.037). Consistent with our findings, pharmacologic inhibition of LDH showed β-catenin mutant cell lines are more dependent on LDH for ATP generation under ischemia than WT cells (EC50: WT: 5.39 vs 4.27 uM, p=0.46, Mut: 12.77 to 0.69 uM (p< 0.0001).

Conclusion: Our findings suggest that HCC tumors with β-catenin pathway mutations have increased dependence on oxidative phosphorylation which renders them more susceptible to TACE-induced ischemia.