Hyperactivation of oxidative mitochondrial metabolism in epithelial cancer cells in situ

Visualizing the therapeutic effects of metformin in tumor tissue

Diana Whitaker-Menezes, Ubaldo E. Martinez-Outschoorn, Neal Flomenberg, Ruth C. Birbe, Agnieszka K. Witkiewicz, Anthony Howell, Stephanos Pavlides, Aristotelis Tsirigos, Adam Ertel, Richard G. Pestell, Paolo Broda, Carlo Minetti, Michael P. Lisanti, Federica Sotgia

Research output: Contribution to journalArticle

141 Citations (Scopus)

Abstract

We have recently proposed a new mechanism for explaining energy transfer in cancer metabolism. In this scenario, cancer cells behave as metabolic parasites, by extracting nutrients from normal host cells, such as fibroblasts, via the secretion of hydrogen peroxide as the initial trigger. Oxidative stress in the tumor microenvironment then leads to autophagy-driven catabolism, mitochondrial dys-function and aerobic glycolysis. This, in turn, produces high-energy nutrients (such as L-lactate, ketones and glutamine) that drive the anabolic growth of tumor cells, via oxidative mitochondrial metabolism. A logical prediction of this new "parasitic" cancer model is that tumor-associated fibroblasts should show evidence of mitochondrial dys-function (mitophagy and aerobic glycolysis). In contrast, epithelial cancer cells should increase their oxidative mitochondrial capacity. To further test this hypothesis, here we subjected frozen sections from human breast tumors to a staining procedure that only detects functional mitochondria. This method detects the in situ enzymatic activity of cytochrome C oxidase (COX), also known as Complex IV. Remarkably, cancer cells show an over-abundance of COX activity, while adjacent stromal cells remain essentially negative. Adjacent normal ductal epithelial cells also show little or no COX activity, relative to epithelial cancer cells. Thus, oxidative mitochondrial activity is selectively amplified in cancer cells. Although COX activity staining has never been applied to cancer tissues, it could now be used routinely to distinguish cancer cells from normal cells, and to establish negative margins during cancer surgery. Similar results were obtained with NADH activity staining, which measures Complex I activity, and succinate dehydrogenase (SDH) activity staining, which measures Complex II activity. COX and NADH activities were blocked by electron transport inhibitors, such as Metformin. This has mechanistic and clinical implications for using Metformin as an anti-cancer drug, both for cancer therapy and chemo-prevention. We also immuno-stained human breast cancers for a series of well-established protein biomarkers of metabolism. More specifically, we now show that cancer-associated fibroblasts overexpress markers of autophagy (cathepsin B), mitophagy (BNIP3L) and aerobic glycolysis (MCT4). Conversely, epithelial cancer cells show the overexpression of a mitochondrial membrane marker (TO MM20), as well as key components of Complex IV (MT-CO1) and Complex II (SDH-B). We also validated our observations using a bioinformatics approach with data from >2,000 breast cancer patients, which showed the transcriptional upregulation of mitochondrial oxidative phosphorylation (OXPHOS ) in human breast tumors (p < 10 -20), and a specific association with metastasis. Therefore, upregulation of OXPHOS in epithelial tumor cells is a common feature of human breast cancers. In summary, our data provide the first functional in vivo evidence that epithelial cancer cells perform enhanced mitochondrial oxidative phosphorylation, allowing them to produce high amounts of ATP. Thus, we believe that mitochondria are both the "powerhouse" and "Achilles' heel" of cancer cells.

Original languageEnglish (US)
Pages (from-to)4047-4064
Number of pages18
JournalCell Cycle
Volume10
Issue number23
DOIs
StatePublished - Dec 1 2011

Fingerprint

Metformin
Therapeutic Uses
Epithelial Cells
Neoplasms
Breast Neoplasms
Oxidative Phosphorylation
Glycolysis
Mitochondrial Degradation
Staining and Labeling
Oxidoreductases
Succinate Dehydrogenase
Autophagy
Mitochondria
Up-Regulation
Electron Transport Complex I
Food
Cathepsin B
Tumor Microenvironment
Energy Transfer
Frozen Sections

Keywords

  • Aerobic glycolysis
  • Autophagy
  • Cancer metabolism
  • Complex I
  • Complex IV
  • Cytochrome c oxidase (COX)
  • Electron transport
  • Metformin
  • Mitochondria
  • Mitophagy
  • NADH dehydrogenase
  • Oxidative phosphorylation (OXPHOS)
  • Respiratory chain
  • Warburg effect
  • Warburg's respiratory enzyme

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology
  • Developmental Biology

Cite this

Hyperactivation of oxidative mitochondrial metabolism in epithelial cancer cells in situ : Visualizing the therapeutic effects of metformin in tumor tissue. / Whitaker-Menezes, Diana; Martinez-Outschoorn, Ubaldo E.; Flomenberg, Neal; Birbe, Ruth C.; Witkiewicz, Agnieszka K.; Howell, Anthony; Pavlides, Stephanos; Tsirigos, Aristotelis; Ertel, Adam; Pestell, Richard G.; Broda, Paolo; Minetti, Carlo; Lisanti, Michael P.; Sotgia, Federica.

In: Cell Cycle, Vol. 10, No. 23, 01.12.2011, p. 4047-4064.

Research output: Contribution to journalArticle

Whitaker-Menezes, D, Martinez-Outschoorn, UE, Flomenberg, N, Birbe, RC, Witkiewicz, AK, Howell, A, Pavlides, S, Tsirigos, A, Ertel, A, Pestell, RG, Broda, P, Minetti, C, Lisanti, MP & Sotgia, F 2011, 'Hyperactivation of oxidative mitochondrial metabolism in epithelial cancer cells in situ: Visualizing the therapeutic effects of metformin in tumor tissue', Cell Cycle, vol. 10, no. 23, pp. 4047-4064. https://doi.org/10.4161/cc.10.23.18151
Whitaker-Menezes, Diana ; Martinez-Outschoorn, Ubaldo E. ; Flomenberg, Neal ; Birbe, Ruth C. ; Witkiewicz, Agnieszka K. ; Howell, Anthony ; Pavlides, Stephanos ; Tsirigos, Aristotelis ; Ertel, Adam ; Pestell, Richard G. ; Broda, Paolo ; Minetti, Carlo ; Lisanti, Michael P. ; Sotgia, Federica. / Hyperactivation of oxidative mitochondrial metabolism in epithelial cancer cells in situ : Visualizing the therapeutic effects of metformin in tumor tissue. In: Cell Cycle. 2011 ; Vol. 10, No. 23. pp. 4047-4064.
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T1 - Hyperactivation of oxidative mitochondrial metabolism in epithelial cancer cells in situ

T2 - Visualizing the therapeutic effects of metformin in tumor tissue

AU - Whitaker-Menezes, Diana

AU - Martinez-Outschoorn, Ubaldo E.

AU - Flomenberg, Neal

AU - Birbe, Ruth C.

AU - Witkiewicz, Agnieszka K.

AU - Howell, Anthony

AU - Pavlides, Stephanos

AU - Tsirigos, Aristotelis

AU - Ertel, Adam

AU - Pestell, Richard G.

AU - Broda, Paolo

AU - Minetti, Carlo

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AB - We have recently proposed a new mechanism for explaining energy transfer in cancer metabolism. In this scenario, cancer cells behave as metabolic parasites, by extracting nutrients from normal host cells, such as fibroblasts, via the secretion of hydrogen peroxide as the initial trigger. Oxidative stress in the tumor microenvironment then leads to autophagy-driven catabolism, mitochondrial dys-function and aerobic glycolysis. This, in turn, produces high-energy nutrients (such as L-lactate, ketones and glutamine) that drive the anabolic growth of tumor cells, via oxidative mitochondrial metabolism. A logical prediction of this new "parasitic" cancer model is that tumor-associated fibroblasts should show evidence of mitochondrial dys-function (mitophagy and aerobic glycolysis). In contrast, epithelial cancer cells should increase their oxidative mitochondrial capacity. To further test this hypothesis, here we subjected frozen sections from human breast tumors to a staining procedure that only detects functional mitochondria. This method detects the in situ enzymatic activity of cytochrome C oxidase (COX), also known as Complex IV. Remarkably, cancer cells show an over-abundance of COX activity, while adjacent stromal cells remain essentially negative. Adjacent normal ductal epithelial cells also show little or no COX activity, relative to epithelial cancer cells. Thus, oxidative mitochondrial activity is selectively amplified in cancer cells. Although COX activity staining has never been applied to cancer tissues, it could now be used routinely to distinguish cancer cells from normal cells, and to establish negative margins during cancer surgery. Similar results were obtained with NADH activity staining, which measures Complex I activity, and succinate dehydrogenase (SDH) activity staining, which measures Complex II activity. COX and NADH activities were blocked by electron transport inhibitors, such as Metformin. This has mechanistic and clinical implications for using Metformin as an anti-cancer drug, both for cancer therapy and chemo-prevention. We also immuno-stained human breast cancers for a series of well-established protein biomarkers of metabolism. More specifically, we now show that cancer-associated fibroblasts overexpress markers of autophagy (cathepsin B), mitophagy (BNIP3L) and aerobic glycolysis (MCT4). Conversely, epithelial cancer cells show the overexpression of a mitochondrial membrane marker (TO MM20), as well as key components of Complex IV (MT-CO1) and Complex II (SDH-B). We also validated our observations using a bioinformatics approach with data from >2,000 breast cancer patients, which showed the transcriptional upregulation of mitochondrial oxidative phosphorylation (OXPHOS ) in human breast tumors (p < 10 -20), and a specific association with metastasis. Therefore, upregulation of OXPHOS in epithelial tumor cells is a common feature of human breast cancers. In summary, our data provide the first functional in vivo evidence that epithelial cancer cells perform enhanced mitochondrial oxidative phosphorylation, allowing them to produce high amounts of ATP. Thus, we believe that mitochondria are both the "powerhouse" and "Achilles' heel" of cancer cells.

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KW - Complex IV

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KW - Metformin

KW - Mitochondria

KW - Mitophagy

KW - NADH dehydrogenase

KW - Oxidative phosphorylation (OXPHOS)

KW - Respiratory chain

KW - Warburg effect

KW - Warburg's respiratory enzyme

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