by ecancer reporter Clare Sansom

Very few cancer patients are killed by their primary tumours; rather, almost all cancer deaths are due to metastatic disease.

Metastatic tumours therefore represent the primary challenge for the development of cancer therapeutics.

We know that both the innate and the adaptive immune systems are involved in modulating tumour growth and metastasis, and that in some circumstances it is possible to induce the immune system to attack tumour cells.

Natural killer cells (NK cells) are cytotoxic lymphocytes that can be activated to respond rapidly to viral infection or tumour formation, triggering apoptosis in the infected or cancerous cell; they form a critical part of the innate immune system.

A large, international group of researchers led by Josef Penninger of the Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria, has now shown that it is possible to use small molecules to activate NK cells to attack cancer metastases.

Genetic knockdown or functional inactivation of an E3 ubiquitin ligase known as Cbl-b is known to activate the cytotoxic CD8 T cells of the adaptive immune system to attack primary tumour cells.

This protein is normally expressed in NK cells and its loss of expression or activity has no effect on these cells’ development.

Penninger and his co-workers first showed that NK cells that had lost Cbl-b activity proliferated more readily; they also secreted more gamma interferon (IFN-γ) and killed NK cell target tumour cell lines more efficiently than similar cells with normal levels of Cbl-b activity.

They then explored whether loss of this protein could activate NK cells specifically against metastatic tumour cells, and found that lung cancer bearing mice in which the Cbl-b gene had been removed or the protein product inactivated developed fewer metastases and survived longer compared to similar mice expressing functional Cbl-b.

Depletion of NK cells in these mice caused uncontrollable tumour growth and metastasis, implicating these cells in the anti-metastatic response.

This finding was confirmed by transplanting NK cells with or without functional Cbl-b into mice bearing lung tumours, and showing that those that received NK cells without the functional protein developed fewer metastases.

The function of ubiquitin ligase is to tag specific proteins for degradation by the covalent attachment of a small protein called ubiquitin to a lysine residue on the target protein.

Penninger and his co-workers set out to discover whether inactivating the protein substrates of Cbl-b would have a similar anti-metastatic effect to inactivating Cbl-b itself.

Using a screen of 9,000 human proteins, they showed that the proteins that were principally ubiquitinylated (and thus targeted for degradation) by Cbl-b were a family of cell-surface tyrosine kinase receptors known collectively as TAM.

The researchers developed a potent and selective small molecule inhibitor of the TAM kinases they termed LDC1267.

This molecule enhanced the cytotoxicity of wild type NK cells towards metastatic tumour cells but had no effect on the already enhanced cytotoxicity of NK cells lacking functional Cbl-b protein.

To confirm that LDC1267 could, indeed, provide NK cells with “licence to kill” metastatic tumour cells, the researchers injected melanoma-bearing mice with this compound and found that it significantly reduced metastatic spread but only in mice with normal quantities of wild type NK cells.

Similar results were obtained in a mouse model of breast cancer, where the numbers and sizes of lung metastases were reduced by intraperitoneal treatment with LDC1267, and the compound was also shown to be effective when administered orally.

The widely used anticoagulant, warfarin, is known to reduce metastasis in rodent models of cancer, and there has been much speculation about its mechanism of action.

However, warfarin is also known to prevent the activation of TAM kinases, and Penninger and his colleagues proposed that it might act on the same pathway to activate NK cells to attack metastases.

They tested this hypothesis by administering low doses of warfarin to melanoma-bearing mice, and found that its anti-metastatic effect was dependent on both Cbl-b activity and the presence of NK cells.

Taken together, these results solve the long-standing mystery of the mechanism of action of warfarin in reducing metastasis, and suggest that it might be possible to develop a drug that would, through inhibiting TAM kinase activity, activate a patient’s NK cells to kill metastases.

Reference

Paolino, M., Choidas, A., Wallner, S. and 21 others (2014). The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells. Nature, published online ahead of print 19 February 2014. doi:10.1038/nature12998