Something else also happens when dividing cells are treated with 5-azacytidine. We now know that when DNMT1 binds at a region where the DNA contains 5-azacytidine instead of the normal cytidine, the DNMT1 becomes stuck there[172]. This marooned enzyme is then sent to a different part of the cell and is broken down. Because of this, the total levels of DNMT1 enzyme in the cell fall[173][174]. The combination of this decrease in the amount of DNMT1, and the fact that 5-azacytidine can’t be methylated, means that the amount of DNA methylation in the cell keeps dropping. We’ll come back in a little while to why this drop in DNA methylation has an anti-cancer effect.
So, 5-azacytidine is an example of where an anti-cancer agent was unexpectedly shown to work epigenetically. Bizarrely, a rather similar thing happened with our second example of a compound which is now licensed to treat cancer[175].
In 1971 the scientist Charlotte Friend showed that a very simple compound called DMSO (its full name is dimethyl sulfoxide) had an odd effect on the cancer cells from a mouse model of leukaemia. When these cells were treated with DMSO, they turned red. This was because they had switched on the gene for haemoglobin, the pigment that gives red blood cells their colour[176]. Leukaemia cells normally never switch on this gene and the mechanism behind this effect of DMSO was completely unknown.
Ronald Breslow at Columbia University and Paul Marks and Richard Rifkind at Memorial Sloan-Kettering Cancer Center were intrigued by Charlotte Friend’s research. Ronald Breslow began to design and create a new set of chemicals, using the structure of DMSO as his starting point, and then adding or changing bits, a little like making new combinations of Lego bricks. Paul Marks and Richard Rifkind began to test these chemicals in various cell models. Some of the compounds had a different effect from DMSO. They stopped cells from growing.
After many iterations, learning from each new and more complicated set of structures, the scientists created a molecule called SAHA (suberoylanilide hydroxamic acid). This compound was really effective at stopping growth and/or causing cell death in cancer cell lines[177]. However, it was another two years before the team were able to identify what SAHA was doing in cells. The key moment happened more than 25 years after Charlotte Friend’s breakthrough publication, when Victoria Richon in Paul Marks’ team, read a 1990 paper from a group at the University of Tokyo.
The Japanese group had been working on a compound called Trichostatin A or TSA. TSA was known to be able to stop cells proliferating. The Japanese group showed that treatment with TSA altered the extent to which histone proteins are decorated with the acetyl chemical group in cancer cell lines. Histone acetylation is another epigenetic modification that we first met in Chapter 4. When cells were treated with TSA, the levels of histone acetylation went up. This wasn’t because the compound was activating the enzymes that put the acetyl groups on histones. It was because TSA was inhibiting the enzymes that remove acetyl groups from these chromatin proteins. These proteins are called histone deacetylases, or HDACs for short[178].
Victoria Richon compared the structure of TSA with the structure of SAHA, and the two are shown in Figure 11.2.
Figure 11.2 The structures of TSA and SAHA, with the areas of greatest similarity circled. C: carbon; H: hydrogen; N: nitrogen; O: oxygen. For simplicity, some carbon atoms have not been explicitly shown, but are present where there is a junction of two lines.
You don’t need a chemistry degree to see that TSA and SAHA look fairly similar, especially at the right hand side of each molecule. Victoria Richon hypothesised that, just like TSA, SAHA was also an HDAC inhibitor. In 1998, she and her colleagues published a paper that showed this was indeed the case[179]. SAHA prevents HDAC enzymes from removing acetyl groups from histone proteins, and as a result, the histones carry lots of acetyl groups.
So, 5-azacytidine and SAHA both decrease cancer cell proliferation, and both inhibit the activity of epigenetic enzymes. Although we could take this as promising support for the theory that epigenetic proteins are important in cancer, perhaps we could just be leaping to conclusions? It might just be a coincidence that both drugs affect epigenetic proteins. After all, the enzymes targeted by the two compounds are very different. 5-azacytidine inhibits the DNMT enzymes, which add methyl groups to DNA. SAHA, on the other hand, inhibits the HDAC family of enzymes, which remove acetyl groups from histone proteins. Superficially, these seem like very different processes. Maybe it’s just coincidence that both 5-azacytidine and SAHA inhibit epigenetic enzymes?
Epigeneticists believe that it is far from being a coincidence. DNA methyltransferase enzymes add a methyl group to the cytidine base. High concentrations of this base are found in the long CG-rich stretches of DNA known as CpG islands. These islands are found upstream of genes, in the promoter regions that control gene expression. When the DNA of a CpG island is heavily methylated, the gene controlled by that promoter is switched off. In other words, DNA methylation is a repressive modification. DNMT activity increases DNA methylation and therefore represses gene expression. By inhibiting these enzymes with 5-azacytidine, we can drive gene expression up.
Histone proteins are also found at the promoters of genes. Histone modifications can be very complex, as we saw in Chapter 4. But histone acetylation is the most straightforward in terms of its effects on gene expression. If the histones upstream of a gene are heavily acetylated, the gene is likely to be highly expressed. If the histones are lacking acetylation, the gene is likely to be switched off. Histone deacetylation is a repressive change. Histone deacetylases (HDACs) remove the acetyl groups from histone proteins and will therefore repress gene expression. By inhibiting these enzymes with SAHA, we can drive gene expression up.
So there is a consistent finding. Our two unrelated compounds, which control growth of cancer cells in culture and which have now been licensed for use in human treatment, inhibit epigenetic enzymes. In doing so, they both drive up gene expression which raises the obvious question of why this is useful for treating cancer. To understand this, we need to get to grips with some cancer biology.
Cancer is the result of abnormal and uncontrolled proliferation of cells. Normally, the cells of our body divide and proliferate at exactly the right rate. This is controlled by a complex balancing act between networks of genes in our cells. Certain genes promote cell proliferation. These are sometimes referred to as proto-oncogenes. They were represented by a plus sign in the see-saw diagram in the previous chapter. Other genes hold the cell back, preventing too much proliferation. These genes are called tumour suppressors. They were represented by a negative sign on the same diagram.
Proto-oncogenes and tumour suppressors are not intrinsically good or bad. In healthy cells, the activities of these two classes of genes balance each other. But when regulation of these networks goes wrong, cell proliferation may become mis-regulated. If a proto-oncogene becomes over-active, it may push a cell towards a cancerous state. Conversely, if a tumour suppressor gets inactivated, it will no longer act as a brake on cell division. The outcome is the same in both cases – the cell may begin to proliferate too rapidly.
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For an excellent history of the development of SAHA, see Marks and Breslow (2007),