Smoking and biological sex shape healthy bladder tissue evolution, offering clues to cancer risk

Bladder cancer is one of the most common cancers worldwide. Men are around four times more likely to develop it than women, and smoking is the main known environmental risk factor. However, the biological mechanisms behind these risk factors remain unclear. Since cancer can take decades to develop, it is important to look at healthy tissues to understand the very first steps of the disease, with the goal of improving risk prediction, prevention, and early diagnosis.

Researchers led by Dr. Núria López-Bigas and Dr. Abel González-Pérez at IRB Barcelona, and Dr. Rosana Risques at the University of Washington, have now shown that smoking and biological sex influence how normal cells evolve in healthy bladder tissue, favouring the expansion of some mutated cells, which may be key in the development of cancer. Published in the journal Nature, the work could provide insights into why men and smokers are at higher risk of bladder cancer. The research was delivered in part through team PROMINENT, as part of the prestigious Cancer Grand Challenges initiative, to answer fundamental questions surrounding early tumour development and identify new opportunities for intervention and routes to cancer prevention.

“Healthy tissues accumulate many mutations throughout life, but what matters is not only how many there are, but which ones manage to outgrow the others and expand into clones, which are copies of the same cell carrying the same mutations,” explains Dr. López-Bigas, ICREA researcher and leader of the Biomedical Genomics group at IRB Barcelona, and Co-lead of Team PROMINENT, Cancer Grand Challenges. “We have seen that smoking and biological sex directly influence this process.”

Looking at cancer before it appears

Cancer does not arise overnight. Over decades, the cells in a tissue accumulate mutations, and some clones gain an advantage that allows them to grow faster than others. This study shows that such evolutionary differences are already visible in healthy tissue before disease sets in.

To carry out the study, the researchers analysed bladder samples from 45 donors. Using a novel approach, comparable to looking at DNA with a powerful new microscope, they were able to detect and quantify thousands of mutations that are invisible to current DNA sequencing techniques.

“The universe is full of stars, but you can’t see most of them without the right instrument. This approach is like going from a backyard telescope to the James Webb Space Telescope: suddenly, multiple mutations become visible in healthy bladder tissue, long before a tumour exists,” says Dr. Risques, University of Washington, co-senior author of the study.

Findings: how sex and smoking shape healthy bladder tissue

The researchers observed clear biological differences between men and women. In male donors, certain mutations in cancer-related genes showed an evolutionary advantage, meaning that clones carrying them were more likely to expand even in the healthy bladder.

They also saw a striking effect of smoking. Among donors over the age of 55, those with a history of smoking carried a high frequency of mutations in the TERT promoter, a DNA element that reactivates telomerase and allows cells to avoid ageing and continue dividing. Most importantly, the work provides evidence that tobacco is not only a mutagen—causing new mutations—but also clonal promoter, facilitating the expansion of cells with pre-existing mutations.

This is the first time such effects have been observed directly in healthy bladder tissue, rather than in tumours, shedding light on the earliest stages of cancer development.

Implications: new insights for cancer risk and prevention

By showing that differences in mutation expansion are already present in healthy tissue, the study offers a new way to understand tissue evolution and provide hints of its malignant transformation. This shift in focus—from simply counting mutations to asking which ones thrive—could help explain why men and smokers are more likely to develop bladder cancer.

The findings also pave the way for future applications. Measuring expanding clones in the bladder could eventually inform risk prediction and early detection tools, for example, by urine sample analysis. More broadly, the same approach could be extended to other tissues and exposures—including workplace chemicals or chemotherapy— thereby opening up new avenues for cancer prevention.

“This study is only the tip of the iceberg. We analysed 16 genes in the bladders of 45 people, yet we already found important differences. The same approach can be applied to other tissues and risk factors, ” adds Dr. González-Pérez, research associate at IRB Barcelona.

Ferriol Calvet and Raquel Blanco, PhD students at IRB Barcelona’s Biomedical Genomics lab, are the first authors of this study. 

This research was delivered as part of the PROMINENT team supported by the  Cancer Grand Challenges initiative, funded by Cancer Research UK, the US National Cancer Institute (NCI), and the Spanish Association Against Cancer (AECC). The work was also supported by the Department of Laboratory Medicine and Pathology at the University of Washington, and benefited from the UK Biobank resource, and institutional and individual fellowships from the AECC, EMBO, and the Spanish government, among others. Dr. Risques was a consultant and equity holder, received research funding from TwinStrand Biosciences, and is the named inventor on a patent owned by the University of Washington and licensed to TwinStrand Biosciences.

Related article:
Sex and smoking bias in the selection of somatic mutations in human bladder
Ferriol Calvet, Raquel Blanco Martinez-Illescas, Ferran Muiños, Maria Tretiakova, Elena S. Latorre-Esteves, Jeanne Fredrickson, Maria Andrianova, Stefano Pellegrini, Axel Rosendahl Huber, Joan Enric Ramis-Zaldivar, Shuyi Charlotte An, Elana Thieme, Brendan F. Kohrn, Miguel Grau, Abel Gonzalez-Perez, Nuria Lopez-Bigas, Rosa Ana Risques
Nature (2025) DOI: 10.1038/s41586-025-09521-x