Bioenergetic Phenotyping of DEN-Induced Hepatocellular Carcinoma Reveals a Link Between Adenylate Kinase Isoform Expression and Reduced Complex I-Supported Respiration

Kelsey L McLaughlin; Margaret A M Nelson; Hannah S Coalson; James T Hagen; McLane M Montgomery; Ashley R Wooten; Tonya N Zeczycki; Nasreen A Vohra; Kelsey H Fisher-Wellman

doi:10.3389/fonc.2022.919880

Bioenergetic Phenotyping of DEN-Induced Hepatocellular Carcinoma Reveals a Link Between Adenylate Kinase Isoform Expression and Reduced Complex I-Supported Respiration

Front Oncol. 2022 Jun 8:12:919880. doi: 10.3389/fonc.2022.919880. eCollection 2022.

Authors

Kelsey L McLaughlin^{1

2}, Margaret A M Nelson^{1

2}, Hannah S Coalson^{1

2}, James T Hagen^{1

2}, McLane M Montgomery^{1

2}, Ashley R Wooten³, Tonya N Zeczycki³, Nasreen A Vohra⁴, Kelsey H Fisher-Wellman^{1

2

5}

Affiliations

¹ Brody School of Medicine, Department of Physiology, East Carolina University, Greenville, NC, United States.
² East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, NC, United States.
³ Brody School of Medicine, Department of Biochemistry and Molecular Biology, East Carolina University, Greenville, NC, United States.
⁴ Brody School of Medicine, Department of Surgery, East Carolina University, Greenville, NC, United States.
⁵ UNC Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, United States.

Abstract

Hepatocellular carcinoma (HCC) is the most common form of liver cancer worldwide. Increasing evidence suggests that mitochondria play a central role in malignant metabolic reprogramming in HCC, which may promote disease progression. To comprehensively evaluate the mitochondrial phenotype present in HCC, we applied a recently developed diagnostic workflow that combines high-resolution respirometry, fluorometry, and mitochondrial-targeted nLC-MS/MS proteomics to cell culture (AML12 and Hepa 1-6 cells) and diethylnitrosamine (DEN)-induced mouse models of HCC. Across both model systems, CI-linked respiration was significantly decreased in HCC compared to nontumor, though this did not alter ATP production rates. Interestingly, CI-linked respiration was found to be restored in DEN-induced tumor mitochondria through acute in vitro treatment with P1, P5-di(adenosine-5') pentaphosphate (Ap5A), a broad inhibitor of adenylate kinases. Mass spectrometry-based proteomics revealed that DEN-induced tumor mitochondria had increased expression of adenylate kinase isoform 4 (AK4), which may account for this response to Ap5A. Tumor mitochondria also displayed a reduced ability to retain calcium and generate membrane potential across a physiological span of ATP demand states compared to DEN-treated nontumor or saline-treated liver mitochondria. We validated these findings in flash-frozen human primary HCC samples, which similarly displayed a decrease in mitochondrial respiratory capacity that disproportionately affected CI. Our findings support the utility of mitochondrial phenotyping in identifying novel regulatory mechanisms governing cancer bioenergetics.

Keywords: bioenergetics; cancer; hepatocellular carcinoma; metabolism; mitochondria.