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

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

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

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

Mitochondrial alterations accompany forced differentiation in acute myeloid leukemia.

Leukemia is characterized by blocked hematopoietic differentiation. Blocked differentiation results in the uncontrolled proliferation of immature, malignant myeloblasts. To counteract myeloblast proliferation, forced differentiation has been studied as a possible treatment for various types of leukemia -- most notably, acute promyelocytic leukemia (APL). In APL, standard of care encompasses treatment with all trans retinoic acid (ATRA), administered alongside chemotherapy or arsenic trioxide. Although the combination of ATRA with chemotherapy achieves complete remission in ~ 90% of APL patients, relapse remains a major clinical problem. For example, treatment with ATRA alone induces only temporary remission, with relapse usually occurring within six months. In vitro studies of differentiation in leukemia cells have shown that the cells seem to take on the phenotype of a terminally differentiated cell, but it is unclear whether the biochemical processes intrinsic to the cell return to normal. While changes in mitochondrial content and function are known to occur with hematopoietic differentiation, the impacts of ATRA-induced differentiation on mitochondrial bioenergetics has yet to be explored. To address this gap in knowledge, a human AML-M2 cell line, HL-60, was treated with ATRA, and the resulting changes in proliferation, morphology, and mitochondrial function and protein expression were investigated. In response to ATRA, cellular proliferation halted by day three of treatment and histology confirmed altered morphological appearance, consistent with myeloid differentiation. In treated cells, mitochondrial respiration was decreased; however, mitochondrial content was unchanged. Interestingly, the respiratory profile of ATRA-differentiated HL60 cells differed substantially from that of healthy primary human granulocytes. These findings were corroborated by differences in size and morphological appearance between the ATRA-treated HL-60 cells and primary human granulocytes. Although further investigation is necessary to fully elucidate the effects of ATRA-induced differentiation of HL-60 cells, these findings suggest that the clinical success of ATRA is likely not the result of APL terminal differentiation

Mitochondrial alterations accompany forced differentiation in acute myeloid leukemia.

Leukemia is characterized by blocked hematopoietic differentiation. Blocked differentiation results in the uncontrolled proliferation of immature, malignant myeloblasts. To counteract myeloblast proliferation, forced differentiation has been studied as a possible treatment for various types of leukemia -- most notably, acute promyelocytic leukemia (APL). In APL, standard of care encompasses treatment with all trans retinoic acid (ATRA), administered alongside chemotherapy or arsenic trioxide. Although the combination of ATRA with chemotherapy achieves complete remission in ~ 90% of APL patients, relapse remains a major clinical problem. For example, treatment with ATRA alone induces only temporary remission, with relapse usually occurring within six months. In vitro studies of differentiation in leukemia cells have shown that the cells seem to take on the phenotype of a terminally differentiated cell, but it is unclear whether the biochemical processes intrinsic to the cell return to normal. While changes in mitochondrial content and function are known to occur with hematopoietic differentiation, the impacts of ATRA-induced differentiation on mitochondrial bioenergetics has yet to be explored. To address this gap in knowledge, a human AML-M2 cell line, HL-60, was treated with ATRA, and the resulting changes in proliferation, morphology, and mitochondrial function and protein expression were investigated. In response to ATRA, cellular proliferation halted by day three of treatment and histology confirmed altered morphological appearance, consistent with myeloid differentiation. In treated cells, mitochondrial respiration was decreased\; however, mitochondrial content was unchanged. Interestingly, the respiratory profile of ATRA-differentiated HL60 cells differed substantially from that of healthy primary human granulocytes. These findings were corroborated by differences in size and morphological appearance between the ATRA-treated HL-60 cells and primary human granulocytes. Although further investigation is necessary to fully elucidate the effects of ATRA-induced differentiation of HL-60 cells, these findings suggest that the clinical success of ATRA is likely not the result of APL terminal differentiation