In a new study published in Nature, researchers from the Wellcome Sanger Institute and collaborators investigated how Notch1 mutants colonize the epithelium, their impact on tissue maintenance, and their effect on oesophageal carcinogenesis. They found that heterozygous Notch1 mutations gives cells an advantage over their neighbours, enabling them to push out other cells and take over normal tissue, as well as impairing the growth of tumours in mice.
A protective mutation?
Oesophageal cancer is the sixth leading cause of cancer deaths globally, with more than 500,000 new cases diagnosed annually. Ageing tissues accumulate somatic mutations, some of which confer a competitive advantage on progenitor cells, forming mutant clones that colonize normal tissue. These clonal expansions are often associated with mutations linked to cancer and may represent the first step in malignant transformation.
Notch1 is a cell surface receptor that plays a role in various cellular processes, including cell proliferation, differentiation, and survival. The function of Notch1 in cancer is complex and still not fully understood. Previous studies have suggested that Notch1 may act as both a tumour suppressor, by inhibiting cell proliferation and promoting differentiation, or a promoter of carcinogenesis through regulation of cell survival, angiogenesis and cell migration. The role of Notch1 in oesophageal carcinogenesis is also still a topic of ongoing research, with some studies suggesting that Notch1 may promote oesophageal carcinogenesis and others suggesting that Notch1 acts as a tumour suppressor.
To investigate the role of Notch1 mutations in the human oesophagus, the researchers visualised clones resolving which Notch1 mutation(s) or copy number alterations they carry. They performed targeted sequencing on histological sections of normal epithelium from elderly donors and found that most samples of normal human oesophagus tissue that contained Notch1 mutant clones carried biallelic alterations that disrupt signalling. The proportion of epithelium with active Notch1 decreased with age, and in older donors Notch1 mutations were common and associated with NICD1 loss.
Using a transgenic mouse model, the researchers also found that Notch1 mutations increase clonal fitness by altering the cellular fate of progenitor cells in the basal layer and leading to excessive production of progenitor cells, driving mutant clone growth (Figure 1). They also found that areas of epithelium without Notch1 protein progressively increased in size with age, suggesting that Notch1 mutants colonize the ageing mouse oesophagus. The researchers then performed bulk and single-cell RNA sequencing found that there were no substantial differences cell state, cell cycling or cell proliferation between cells expressing wild-type vs mutant Notch1. The study concludes that once cells expressing mutant Notch1 have occupied the epithelium, their behaviour is similar to wild-type cells.
Finally, the study explored the role of Notch1 in oesophageal carcinogenesis by treating mice with a drug (mutagen) that generates dysplastic lesions. The study found that Notch1 wild-type cells are more likely to contribute to tumours than those carrying Notch1 mutations, and also found that wild-type Notch1 favours tumour growth and that blocking Notch1 function reduced tumour size.
Implications and future directions
The results of this study showed that mutations that reduce the function of Notch1 confer a competitive advantage on mutant progenitors, leading to the formation of persistent, expanding clones in the normal oesophageal epithelium. This mechanism explains the disparity in the prevalence of NOTCH1 mutations in normal oesophageal epithelium and tumours.
As the heterozygous mutant population grows, the probability that the remaining allele will be lost increases, further driving the colonization of the epithelium. The study also found that once an area has been colonized by biallelic Notch1 mutants, the phenotype of mutant cells reverts toward that of wild-type cells, which explains the normal appearance of aged human oesophageal epithelium despite NOTCH1 signalling being disrupted in most of the tissue. The study also found that Notch1 loss slows the cell division rate and therefore tumours with biallelic Notch1 disruption are smaller. This research has important implications for the potential use of NOTCH1 inhibitors in reducing the growth of premalignant tumours in the oesophagus.