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Differential squamous cell fates elicited by NRF2 gain-of-function versus KEAP1 loss-of-function

ABSTRACT: Clinical evidence has revealed that high-level activation of NRF2 caused by somatic mutations in NRF2 is frequently detected in esophageal squamous cell carcinoma (ESCC), whereas that by somatic mutations in KEAP1, a negative regulator of NRF2, is not. Here, we challenged to generate a mouse model of NRF2-activated ESCC using the cancer-derived NRF2L30F mutation and cancer-driver mutant Trp53R172H. Concomitant expression of NRF2L30F and Trp53R172H induced proliferation of squamous cell epithelia and resulted in NRF2-activated ESCC-like lesions. In contrast, while squamous cell-specific deletion of KEAP1 induced similar NRF2 hyper-activation, the loss-of-KEAP1 combined with Trp53R172H did not elicit the proliferation and formation of ESCC-like lesions. Instead, KEAP1-deleted cells disappeared from the esophageal epithelium over time by cell competition. These findings provide insights into the observation that somatic mutations are more frequently observed in NRF2 than in KEAP1, and the mouse model developed here will be instrumental in elucidating the mechanistic basis leading to NRF2-activated ESCC.

ORGANISM(S): Mus musculus

PROVIDER: GSE255927 | GEO | 2024/03/22

REPOSITORIES: GEO

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Project description:Esophageal cancer is one of the most common cancers where TP53 is mutated frequently. Esophageal squamous cell carcinoma (ESCC) is the major subtype of esophageal cancer, and is one of the most lethal cancers worldwide. ESCC evolves often to lung metastasis, which is associated with a dismal prognosis. P53R175H (homologous to Trp53R172H in mice) is a common hot spot mutation. How metastasis is regulated by p53R175H in ESCC, or cancers in general, remains to be elucidated. To investigate p53R175H mediated molecular mechanisms, we utilized germline Trp53R172H/- mice, and generated esophageal specific Trp53-/- mice and Trp53+/+ mice treated with the 4-NQO esophageal-specific carcinogen (a common exposure in humans) to model ESCC. In the primary Trp53R172H/- tumor cell lines established from these mouse models, we depleted Trp53R172H (shTrp53) and observed a marked reduction in cell invasion in vitro and lung metastasis burden in a tail-vein injection model in comparing isogenic cells (shCtrl). Furthermore, to understand the molecular basis for mutant p53 driven lung metastasis, we performed bulk RNA-seq to compare gene expression profiles of metastatic and primary shCtrl and shTrp53 cells. We performed Gene Set Enrichment Analysis (GSEA) and identified the YAP-BIRC5 axis as a potential mediator of Trp53R172H mediated metastasis. As a target gene of YAP, BIRC5 encodes an anti-apoptotic protein, namely survivin. We demonstrate that survivin expression increases in the presence of Trp53R172H but not wild-type Trp53. Furthermore, depletion of survivin decreases Trp53R172H driven lung metastasis. Mechanistically, Trp53R172H but not wild-type Trp53, binds with YAP in ESCC cells, suggesting their cooperation to induce survivin expression. Furthermore, survivin high expression level is associated with increased metastasis in several GI cancer, suggesting that survivin may be a key regulator to metastasis. Taken together, this study unravels new insights into how mutant p53 mediates metastasis.

Project description:TP53 mutations are frequent in esophageal squamous cell carcinoma (ESCC), and other SCCs, and are associated with a proclivity for metastasis. Here, we report that the Colony-Stimulating Factor-1 (CSF-1) expression is upregulated significantly in a p53-R175H (R172H in mice)-mediated manner in lung metastatic lesions of ESCC. The p53-R172H-dependent Csf-1 signaling through its cognate receptor Csf-1r increases tumor cell invasion and lung metastasis, which is mediated in part through the phosphorylation of Stat3 and epithelial-to-mesenchymal transition. In Trp53R172H tumor cells, p53 binds to the Csf-1 promoter and the Csf-1 locus is enriched with H3K27ac, which likely is permissive for fostering an interaction between Brd4 and p53-R172H to regulate the Csf-1 transcription. Inhibition of Brd4 not only reduces tumor invasion and lung metastasis but also reduces the Csf-1 level in circulation. Overall, our results establish that the p53-R175H-mediated Brd4-Csf-1 axis promotes ESCC lung metastasis and suggest avenues for therapeutic strategies for this difficult to treat disease. To elucidate the mechanism for how Trp53R172H regulates Csf-1 transcription, we conducted Cleavage Under Targets and Release Using Nuclease sequencing (CUT&RUN-seq) for histone 3 lysine 27 acetylation (H3K27ac), which is an active enhancer and promoter marker [48], on Trp53R172H/- or Trp53+/+ ESCC cells. The genome-wide analysis revealed 1189 genes having enriched and 1454 genes having depleted H3K27ac in Trp53R172H/- tumor cells compared to Trp53+/+ cells (|log2fold change| > 1.5). Notably, H3K27ac peaks were enriched across Vim gene encoding the mesenchymal marker Vimentin and were reduced across Cdh1 gene encoding the epithelial marker E- Cadherin in Trp53R172H tumor cells compared to Trp53+/+ cells. Interestingly, H3K27ac peaks were enriched across Csf-1 locus including the promoter site in Trp53R172H/- ESCC cells compared to Trp53+/+ tumor cells, suggesting that the Csf-1 locus was accessible and active in the presence of p53-R172H. The epigenetic reader Brd4 binds to acetylated histone lysine residues at promoters and super-enhancers to regulate transcription initiation [29]. We therefore investigated if Brd4 was recruited to enriched H3K27ac sites in ESCC cells to upregulate Csf-1. We found that global Brd4 level in Trp53R172H/- tumor cells was ~2-fold higher than in Trp53-/- cells and ~3-fold higher than in Trp53+/+ cells suggesting gain-of-function properties of p53-R172.

Project description:Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers with high mortality rate. Smoking is one of the established risk factors of ESCC. However, there is limited data on molecular alterations associated with cigarette smoke exposure in esophageal cells. Understanding the effects of cigarette smoke on esophageal squamous epithelial cells at a molecular level would lead to a better understanding of the pathobiology of ESCC which has implications for identification of early biomarkers and therapeutic targets. To investigate the effect of cigarette smoke exposure, we developed a cell line model where Het1A cells (non-neoplastic human esophageal epithelial cells) were chronically treated with cigarette smoke condensate (CSC) for 2 months, 4 months, 6 months and 8 months. We carried out comparative proteomic, phosphoproteomic and whole exome sequencing analyses on CSC treated and untreated cells. Increased cell proliferation and invasion of Het1A cells was observed after chronic exposure to cigarette smoke. Using quantitative proteomic and phosphoproteomic analyses, we identified 56 proteins and 296 phosphoproteins that showed differential expression. Bioinformatics analysis of differentially expressed proteins and phosphoproteins showed enrichment of molecules involved in DNA damage response pathway. Whole exome sequencing (WES) of CSC treated and untreated cells also revealed mutations and copy number alterations in genes associated with DNA damage response. By correlating WES, proteomic and phosphoproteomic results, we observed potential loss of function in HMGN2 and MED1 that were reported as potential tumor suppressors and are known to play important role in DNA damage response. We also observed decreased expression of HMGN2 in tissue section of ESCC. Overexpression of HMGN2 and MED1 lead to decreased proliferative and invasive ability of CSC treated cells. These findings suggest that cigarette smoke affects genes and proteins associated with DNA damage response pathways which might play a vital role in development of ESCC.

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Differential squamous cell fates elicited by NRF2 gain-of-function versus KEAP1 loss-of-function

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