by ecancer reporter Clare Sansom

Cancer cells use and metabolise extracellular nutrients in different ways from normal cells, simply because the increased cellular proliferation that drives these cells demands a larger supply of these nutrients.

A full understanding of these differences may lead to the development of novel strategies for suppressing the growth of tumours.

One of the most important of these nutrients is glucose, which is necessary for the metabolism of a large number of molecules that are essential for cell growth.

The increased uptake of glucose by cancer cells is utilised in the clinic in tools for tumour imaging and diagnosis.

The final step of glycolysis – the pathway through which glucose is converted into pyruvate, releasing free energy – is catalysed by the enzyme pyruvate kinase.

Mammals have a total of four isoforms of this enzyme; the Pkm gene encodes two of these, PKM1 and PKM2. PKM1 is constitutively expressed in most normal tissues, whereas PKM2 is expressed in proliferating cells including cancer cells.

This isoform is less active constitutively but can be activated by fructose -1,6-bisphosphate (FBP), a glycolytic metabolite. The active form of both isoforms is known to be a tetramer.

Low PKM2 activity and increased glucose uptake both facilitate the synthesis of more complex molecules from glycolytic products.

A large group of researchers led by Matthew Vander Heiden of Massachusetts Institute of Technology, MA, USA, have now shown that synthetic molecules that activate PKM2 can suppress this anabolism and thence impede the growth of tumours.

Vander Heiden and his co-workers had previously shown that replacing PKM2 by the constitutively active PKM1 could suppress tumour growth in a mouse xenograft model.

They now found that engineered PKM1 expression alone did not affect PKM2 levels in these cells, and that expression of both isoforms increased total pyruvate kinase activity. They used immunoprecipitation to show that the isoforms can associate together into active tetramers. Engineered human tumour cells expressing PKM1 as well as PKM2 generated significantly fewer tumours than unmodified cells from the same cell line when injected into immunocompromised mice.

The PKM2 isoform binds to phosphorylated tyrosine residues, and this binding suppresses the activity of the protein by releasing its activator, FBP. Vander Heiden and his co-workers used pervanadate, which increases the amount of phosphotyrosine in proteins by inhibiting tyrosine phosphatases, to investigate this interaction mechanism and determined that pervanadate prevented the formation of PKM2 tetramers.

However, PKM2 activity was not suppressed when pervanadate was added to cancer cell lysates pre-treated with synthetic small molecules that are known to activate PKM2, TEPP-46 and DASA-58. This indicated that these molecules must use a different mechanism from the endogenous FBP to activate the protein.

The researchers then explored the mechanism of PKM2 activation further by crystallising this protein separately with both TEPP-46 and DASA-58. The resulting structures showed that PKM2 crystallised as a tetramer, with each tetramer bound to four molecules of FBP from the bacterial cells that expressed the protein and two molecules of the synthetic activator. The activator molecules were located in a distinctly different binding site from that of the FBP molecules, at the interface between PKM2 subunits. 

These activators were further shown to stabilise the active form of the enzyme in a model in which tetramer formation had been prevented by the mutation of a key lysine residue at the subunit interface into glutamate.

Taken together, these results show that synthetic PKM2 activators can stabilise the active tetrameric form of the enzyme so that it mimics the activity of the constitutive PKM1 isoform.

To determine the extent to which these molecules affect the metabolism and proliferation of cancer cells, the researchers treated cancer cell lines with the small-molecule activators and with PKM1 under normal and hypoxic conditions. Both interventions reduced cell proliferation significantly only under low oxygen conditions.

Mice bearing human xenograft tumours were then treated with oral doses of TEPP-46. Tumours in mice treated with TEPP-46 were significantly smaller and slower growing than those in control mice treated with vehicle; furthermore, PKM2 extracted from tumours in the treated mice was found to be exclusively in the active tetrameric form, whereas very little of the protein extracted from control mouse tumours was found as tetramers. These results indicate that PKM2 activators might be useful as anti-cancer drugs, particularly if they were combined with compounds that promote oxidative stress.

Reference

 Anastasiou, D., Yu3, Y., Israelsen, W.J. and 40 others (2012).  Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis. Nature Chemical Biology, published online ahead of print 26 August 2012. doi: 10.1038/nchembio.1060