Hereditary leiomyomatosis and renal-cell cancer (HLRCC) is a rare genetic disorder characterised by benign skin tumours, uterine fibroids in women, and a greatly increased risk of developing renal cell carcinoma.

It is known to be caused by mutations in the gene FH, which encodes fumarate hydratase, an enzyme in the Krebs or TCA cycle that catalyses the conversion of fumarate into malate.

Affected individuals are born with a mutation in one copy of this gene and acquire mutations in the other via DNA damage.

Blocking fumarate hydrolase function blocks the TCA cycle leading to an accumulation of metabolites within that cycle, particularly fumarate itself.

It was not previously known, however, how cells with no functional enzyme and thus no functional TCA cycle survive.

An international team led by Eyal Gottlieb at the Beatson Institute for Cancer Research, Glasgow, Scotland, UK, has now used a modified mouse kidney cell line to investigate this pathway. A cell line with a double deletion (knockout) of the equivalent mouse gene, Fh1, was obtained from modified, immortalised mouse kidney cells via adenovirus infection.

The researchers measured levels of different TCA cycle metabolites in these cells using gas chromatography–mass spectrometry, and found that more than 100 times as much fumarate accumulated in them than in cells with normal TCA cycles. There was also some increase in succinate levels and decreases in malate and citrate.

To investigate the source of carbon for the TCA cycle, cells with normal TCA activity and Fh1-/- cells were cultured in media containing either 13C glutamine or 13C glucose. In the Fh1-/- cells cultured with labelled glutamine, the 13C atoms were exclusively incorporated into fumarate, indicating both that these cells use glutamine as a sole source of carbon and that the cycle is blocked at this point. The truncation of the TCA pathway would be expected to block the production of mitochondrial NADH, but levels of this metabolite were found to be only slightly reduced, indicating that an alternative pathway for generating NADH must be used.

Gottlieb and his co-workers used a computer modelling approach based on flux balance analysis (FBA), which is now often used in microbial modelling, to reconstruct the genome-scale metabolic networks of Fh1-/- cells and equivalent cells with normal Fh1 activity.

This technique starts with determining sets of relevant genes that are highly expressed in each cell line using microarray analysis, and incorporates known cellular metabolite concentrations and requirements into a model metabolic network.

A total of twenty-four gene deletions were predicted to be synthetic lethal in the Fh1-/- cell line but not in the normal cell line. Eighteen of these are genes in the same pathway: a linear pathway involving the biosynthesis and degradation of haem that starts with glutamine uptake, ends with bilirubin excretion, and synthesises NADH almost as a by-product.

Three of the genes involved, including haem oxygenase 1 (Hmox1), were also found to be upregulated in Fh1-/- cells. Interestingly, the human form of this gene has also been observed to be up-regulated in uterine fibroids from a patient diagnosed with HLRCC.

The researchers validated the activation of the haem biosynthesis pathway in Fh1-/- cells by measuring levels of excreted bilirubin in the surrounding media, and found, as expected, that these cells excreted more bilirubin than those with normal TCA metabolism.

Silencing Hmox1 and inhibiting its activity with the known Hmox inhibitor, zinc protoporphyrin, both significantly reduced the growth of Fh1-/- mouse cells but not that of normal cells. Similar results were observed using a human kidney cancer cell line derived from a HLRCC patient. Taken together, these results indicate that inhibition of Hmox would be likely to selectively kill FH-deficient cancer cells and would thus be a promising target for drugs against HLRCC.

Reference: Frezza, C., Zheng, L., Folger, O. and 15 others (2011). Haem oxygenase is synthetically lethal with the tumour suppressor fumarate hydratase. Nature, published online ahead of print 18 August 2011. doi:10.1038/nature10363