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The gut microbiome switches mutant p53 from tumour-suppressive to oncogenic

TP53, encoding the tumour suppressor protein p53, is the most commonly mutated cancer-associated gene. The six most frequently mutated residues of p53 mostly result in loss-of-function where the mutant p53 protein cannot activate downstream regulators of homeostasis. Some mutant forms of p53 can have a dominant-negative effect over the wild-type, causing tumorigenesis and oncogenic gain of function.

In this study the authors investigated two of the most common mutations, R172H and R270H, in mice (homologues of human R175H and R273H). Mutations were generated by knock-in and assessed in a WNT-driven mouse model of intestinal neoplasia and a gut-inducible deletion of Csnk1a1, encoding Casein kinase 1a (CK1a), which also drives intestinal cancers.

Deletion of Cskn1a1 causes stabilisation of WT and mutant p53 proteins in the gut. Three models were generated comparing PROX1 as an indicator of dysplasia in WT, mutant and no p53 in the gut.

After CK1a deletion, WT mice had moderate epithelial dysplasia whereas p53 KO mice had high-grade dysplasia with increased proliferation throughout the whole gut. When the R172H mutated form was stabilised, colonic and ileal segments of the gut were highly dysplastic and proliferative, however, the duodenum and jejunum demonstrated normal levels of proliferation and minimal dysplasia in villi and shorter jejunal crypts compared to WT p53 and no p53 models.

CK1a deletion in combination with mutant p53 stabilisation was lethal within ten days, which was too early for any discrete tumours to develop.

In mice three days after CK1a deletion, the R172H and R270H mutations had similar levels of high-grade dysplasia in the distal gut. No evidence of hyperproliferation or invasion were observed in the R270H mutation model in the proximal gut. These mutations present in the proximal gut appear to prevent dysplasia due to p53 loss.

No p53 target genes were expressed strongly in jejunum in response to CK1a stabilisation of the mutant p53 proteins ruling out the possibility that transactivational activity was regained.

ChIP-seq analysis confirmed that mutant p53 was not bound to chromatin suggesting a tumour suppressive effect of mutants in the jejunum was not due to canonical p53 activation.

In a second model, the Apc/Min/+ mouse model, mutant p53 R172H resulted in increased adenoma frequency in the colon, but attenuated tumour formation in the proximal gut confirming that p53 mutants suppress tumour formation in the proximal gut but enhance tumorigenesis in the distal gut.

P53-suppressed invasiveness signature genes (PSIS) are the main set of cancer promoting genes switched off by p53. Many of these genes belong to the WNT pathway. In the jejunum of CK1a KO mice, activation of these genes was observed when p53 was KO. R172H mice expressed less WNT genes consistent with p53 tumour suppression.

Conversely, in the ileum and colon, WNT-gene expression was high in the p53 mutant mice with similar levels to the p53 WT KO.

Transcriptional analysis of R172H and p53 KO mice demonstrated that mutant p53 mostly affected WNT gene sets in the jejunal enterocytes. No other pathways were differentially regulated in proximal or distal gut, suggesting WNT suppression by mutant p53 was specific to jejunal enterocytes.

ChIP-seq analysis demonstrated selective disengagement of TCF4 from WNT-responsive elements in the jejunal enterocytes of p53 mutant mice, in ileal enterocytes this remained as normal.

Jejunal and ileal organoids were generated to investigate the mechanism of p53 mutant suppression of WNT signalling. Organoids with p53 KO had high proliferation and hyperactivation of WNT, and failed to differentiate. Organoids from both ileum and jejunum expressing the mutant p53 had normal proliferation and differentiation and displayed moderate WNT activity.

These findings were also confirmed in human organoids expressing the human homologues of these p53 mutations.

The authors hypothesised that the gut microbiome may provide the switch that turns mutant p53 oncogenic in the distal gut. Gut microflora were eliminated with antibiotics and in R172H mice this treatment attenuated the dysplasia observed in the colon and ileum.

Consistent with the previous results, antibiotic treatment decreased proliferation and WNT activation in p53 mutants, suggesting that gut microbiota block the suppression of WNT by mutant p53 promoting tumour formation in the distal gut.

Organoids were challenged with metabolites from major gut microbe families known to have effects on tumorigenesis, such as butyrate, deoxycholate, sulphorafane, ellagic and gallic acid. Morphology, proliferation and WNT activity were used as endpoint assays.

Polyphenols, urolithin B, ellagic acid and gallic acid, caused rounding of organoids similar to WNT active p53 KO organoids. Gallic acid increased proliferation and WNT activity of p53 mutant organoids and the continuous presence of gallic acid was sufficient to maintain the suppression of mutant p53’s anti-tumour properties.

Gallic acid was found at markedly higher levels in the ileum compared to jejunum most likely due to the action of Lactobacillus plantarum and Bacillus subtilis shikimate dehydrogenase (SDH). Abundant copies of the SDH were observed in ileum, with very few in the jejunum and antibiotic treatment eliminated nearly all copies.

Consistent with this data, treatment with antibiotics and supplementation with gallic acid attenuated tumour suppression by mutant p53 in the distal gut. Both ileum and colon became hyperproliferative, dysplastic and WNT target genes became activated.

In this study the authors used two mouse models that expressed WT, no p53 or mutant p53 to analyse the effect of p53 mutants in the gut. They also used organoids and transcriptomic analysis to identify targets of a mutant p53 tumour suppressive effect that was specific to the proximal gut and had an opposing effect in the distal gut, promoting tumour formation. The authors hypothesised that the gut microbiome may be responsible for this switch and identified gallic acid as the potential source of this switching mechanism. They also demonstrated that this was reversible and speculated that dietary management and that gallic acid inhibitors and antagonists may be a potential therapeutic.

Reference

Kadosh, E., Snir-Alkalay, I., Venkatachalam, A. et al. The gut microbiome switches mutant p53 from tumour-suppressive to oncogenic. Nature (2020)
doi: 10.1038/s41586-020-2541-0

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