Early clues from normal tissue: how sex and smoking influence cancer promotion in the bladder

The PROMINENT team is taking on our normal phenotypes challenge and investigating how environmental exposures and risk factors influence cancer development. The team’s work is centred around the promoter hypothesis in which cells accumulate cancer-driving mutations but remain 'normal’. The exposure to a ‘promoting’ stimulus, confers ‘initiated’ cells with a selective advantage allowing them to undergo clonal expansion and progress to malignancy.

As Nuria puts it, “We frame the question using the initiation and promotion model. Initiation refers to cells acquiring mutations, but we know normal tissues are riddled with mutations and most cells remain normal, so how are these normal cells then promoted to become cancer cells?”

The team’s latest work focused on bladder cancer and uncovering why the risk of developing it is four times higher for men than women. The team asked whether there were sex differences in the bladder before cancer develops that could explain this difference in incidence. Nuria reveals, “The answer is yes - we can already see differences in how somatic mutations are selected between males and females.”

The team found that for three genes implicated in bladder cancer (specifically RBM10, CDKN1A and ARID1A), truncating mutations were selected for at a much higher rate in men than in women. Nuria explains, “The mutation rate is very similar between males and females, so it’s not that they happen more, it’s that when they happen, they provide a bigger selective advantage in males, which leads to clonal expansion.” 

Smoking is also a known risk factor for bladder cancer and while tobacco smoke is known to contain mutagens, the team revealed that surprisingly in bladder cancer tobacco is likely acting as a promoter rather than an initiator. The team found no smoking associated mutational signatures but instead that smokers had increased clonal expansions, specifically of cells carrying mutations in the TERT promoter, the regulatory region controlling the expression of telomerase, which allows cells to avoid ageing and continue dividing. The team’s work highlights the multifaceted effects of smoking in increasing cancer risk, beyond causing mutations and reveals its effects on normal tissue. 

The team will continue to work to identify the promoting event that is causing the differences in clonal expansion between the sexes and the underlying mechanisms. It speculates that hormonal differences between men and women could be involved, or external factors that men are more exposed to.  

Key to the team’s findings is its development of ultradeep DNA duplex sequencing. The approach allowed the team to reveal the full spectrum of mutations that occur in normal bladder tissue, not just the most common. The PROMINENT team is now applying ultradeep DNA duplex sequencing across different tissues. This technical advance will allow the team and others to determine which normal cells will become cancerous and which mutations are the first step on the evolutionary pathway to becoming a tumour. 

Nuria comments, “We previously modelled all the different mutations that can occur in cancer genes in silico and predicted computationally which ones could be drivers or passengers. But with this technique we can actually see them in human tissue – it’s a natural experiment. We’re approaching real saturation mutagenesis.” 

Experimental saturation mutagenesis, where each gene is systematically mutated to study the effect of every possible variant, provides deep insights into the functional effect of each mutation. However, it is a time-consuming and expensive task. As Nuria mentioned, her lab previously described in silico saturation mutatagenesis to do this computationally. Now, the PROMINENT team proposes that ultradeep DNA duplex sequencing allows for natural saturation mutagenesis to determine the functional impact of mutations directly in human tissue.  

Through Cancer Grand Challenges team PROMINENT is funded by Cancer Research UK, the National Cancer Institute and the Scientific Foundation of the Spanish Association Against Cancer. 

Article written by Rebecca Eccles with thanks to Nuria Lopez-Bigas.