Head and neck squamous cell carcinoma (HNSCC), the most common form of head and neck cancer, is diagnosed in almost 900 000 patients annually with a mortality rate of approximately 40% within 5 years of diagnosis. Early diagnosis and effective treatment strategies with limited toxicity are urgently awaited. Alterations in cellular metabolism is one of the hallmarks of cancer and could reveal potential diagnostic options and/or therapeutic targets. We have found that HNSCC cells exhibit a remarkable aerobic fermentation, which we termed the ‘super-Warburg effect’. This means that their lactate production was >2.0 times higher than their glucose consumption, while the full fermentation of one molecule of glucose can maximally yield two lactate molecules.

To study the possible origin of this surplus lactate in a systemic context, we used proteomics from HNSCC cell lines to make context-specific HNSCC models from an existing genome-scale reconstruction of human metabolism (Human1). Flux balance analysis and flux variability analysis revealed anaplerotic amino acids – in addition to glucose – as likely sources of extra carbon. These amino acids enter the Krebs cycle and exit again, for instance as malate, to be decarboxylated to pyruvate, and subsequently to lactate. Our model analysis showed glutamine to be by far the most abundant source of extra carbon, and also showed it to be an obligatory substrate for the cells to grow the at the measured rate with the substrates available in the medium, while maintaining super Warburg lactate production. Malic enzyme 1 (ME1) and serine dehydratase (SDH) were indicated as important nodes in this conversion.

Surprisingly, cultured cells did not take up glutamine from the medium as measured by nuclear magnetic resonance spectroscopy. This could be due to the uptake fluxes simply being undetectably low. One factor causing this might be the availability of alternative amino acid sources. A prominent candidate is the medium component, foetal calf serum (FCS), which contains proteins. It has been shown previously that some cancers can take up proteins by macropinocytosis and convert it into lactate.

We adjusted our model to test the hypothesis that albumin – comprising about two-thirds of FCS protein – could reduce the required glutamine uptake to explain the lack of a detectable glutamine uptake flux in the data. However, the amount of albumin approximated to be present in a growth medium containing 5% of FCS is very low and millimolar changes in glutamine were still necessary to account for the growth rate and lactate production in our simulation. We will further investigate the dependency of the cells on proteins in the medium, and analyse possible flux distributions at low uptake of glutamine. We will also manipulate the genes for ME1 and SDH test their importance in the production of lactate from glutamine.