Defining function of wild-type and three patient specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma
Abstract
In embryonal rhabdomyosarcoma (ERMS) and generally in sarcomas, the role of wild-type and loss or gain-of-function TP53 mutations remains largely undefined. Eliminating mutant or restoring wild-type p53 is challenging; nevertheless, understanding p53 variant effects on tumorigenesis remains central to realizing better treatment outcomes. In ERMS, >70% of patients retain wild-type TP53, yet mutations when present are associated with worse prognosis. Employing a kRASG12D-driven ERMS tumor model and tp53 null (tp53-/-) zebrafish, we define wild-type and patient-specific TP53 mutant effects on tumorigenesis. We demonstrate that tp53 is a major suppressor of tumorigenesis, where tp53 loss expands tumor initiation from <35% to >97% of animals. Characterizing three patient-specific alleles reveals that TP53C176F partially retains wild-type p53 apoptotic activity that can be exploited, whereas TP53P153D and TP53Y220C encode two structurally related proteins with gain-of-function effects that predispose to head musculature ERMS. TP53P153D unexpectedly also predisposes to hedgehog expressing medulloblastomas in the kRASG12D-driven ERMS-model.
Data availability
Data sets were submitted to DRYAD, available here: doi.org/10.5061/dryad.zgmsbccb6
-
TP53 Zebrafish DataNCBI Gene Expression Omnibus, GSE213869.
-
TP53 Zebrafish DataDryad Digital Repository, doi:10.5061/dryad.zgmsbccb6.
Article and author information
Author details
Funding
Cancer Prevention and Research Institute of Texas (Training Award,RP 170345)
- Amanda E Lipsitt
St. Baldrick's Foundation
- David S Libich
Welch Foundation
- David S Libich
University of Texas Health Science Center at San Antonio (Greehey Graduate Fellowship in Children's Health)
- Paulomi Modi
Hyundai Hope On Wheels (Young Investigator Grant)
- Amanda E Lipsitt
Max and Minnie Tomerlin Voelcker Fund (Young Investigator Award)
- Myron S Ignatius
Max and Minnie Tomerlin Voelcker Fund (Young Investigator Award)
- David S Libich
University of Texas Health Science Center at San Antonio (Cancer T32 Fellow,T32CA148724)
- Kunal Baxi
University of Texas Health Science Center at San Antonio (Translational Science TL1 Fellow,TL1TR002647)
- Kunal Baxi
Cancer Prevention and Research Institute of Texas (Scholar Grant,RR160062)
- Myron S Ignatius
University of Texas Health Science Center at San Antonio (Greehey Graduate Fellowship in Children's Health)
- Nicole Rae Hensch
National Institutes of Health (R00CA1715184)
- Peter Houghton
- Myron S Ignatius
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Animal studies were approved by the UT Health San Antonio Institutional Animal Care and Use Committee (IACUC) under protocol #20150015AR (mice) and #20170101AR (zebrafish). Zebrafish images were taken with specimens under tricaine anesthesia. Zebrafish tumor extraction was performed by administering high dose tricaine to minimize suffering.
Human subjects: Patient presenting with osteosarcoma signed a Consent to be part of a Repository, Epidemiology of Cancer in Children, Adolescents and Adults. In brief, this allowed for the storage of tissue, cataloging of medical information, and for research to be conducted from collected samples. The study's IRB number is HSC20080057H. Patient was informed of the risks and benefits. The umbrella study covering epidemiological study and patient-derived xenograft generation is IRB approved through UT Health San Antonio.
Reviewing Editor
- Stephen C Ekker, Mayo Clinic, United States
Version history
- Received: March 9, 2021
- Preprint posted: April 22, 2021 (view preprint)
- Accepted: June 1, 2023
- Accepted Manuscript published: June 2, 2023 (version 1)
- Version of Record published: July 5, 2023 (version 2)
Copyright
© 2023, Chen et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 678
- Page views
-
- 117
- Downloads
-
- 1
- Citations
Article citation count generated by polling the highest count across the following sources: PubMed Central, Crossref, Scopus.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Cancer Biology
Cancer stem cells (CSCs) undergo epithelial-mesenchymal transition (EMT) to drive metastatic dissemination in experimental cancer models. However, tumour cells undergoing EMT have not been observed disseminating into the tissue surrounding human tumour specimens, leaving the relevance to human cancer uncertain. We have previously identified both EpCAM and CD24 as CSC markers that, alongside the mesenchymal marker Vimentin, identify EMT CSCs in human oral cancer cell lines. This afforded the opportunity to investigate whether the combination of these three markers can identify disseminating EMT CSCs in actual human tumours. Examining disseminating tumour cells in over 12,000 imaging fields from 74 human oral tumours, we see a significant enrichment of EpCAM, CD24 and Vimentin co-stained cells disseminating beyond the tumour body in metastatic specimens. Through training an artificial neural network, these predict metastasis with high accuracy (cross-validated accuracy of 87-89%). In this study, we have observed single disseminating EMT CSCs in human oral cancer specimens, and these are highly predictive of metastatic disease.
-
- Cancer Biology
- Medicine
Esophageal cancer (EC) is a fatal digestive disease with a poor prognosis and frequent lymphatic metastases. Nevertheless, reliable biomarkers for EC diagnosis are currently unavailable. Accordingly, we have performed a comparative proteomics analysis on cancer and paracancer tissue-derived exosomes from eight pairs of EC patients using label-free quantification proteomics profiling and have analyzed the differentially expressed proteins through bioinformatics. Furthermore, nano-flow cytometry (NanoFCM) was used to validate the candidate proteins from plasma-derived exosomes in 122 EC patients. Of the 803 differentially expressed proteins discovered in cancer and paracancer tissue-derived exosomes, 686 were up-regulated and 117 were down-regulated. Intercellular adhesion molecule-1 (CD54) was identified as an up-regulated candidate for further investigation, and its high expression in cancer tissues of EC patients was validated using immunohistochemistry, real-time quantitative PCR (RT-qPCR), and western blot analyses. In addition, plasma-derived exosome NanoFCM data from 122 EC patients concurred with our proteomic analysis. The receiver operating characteristic (ROC) analysis demonstrated that the AUC, sensitivity, and specificity values for CD54 were 0.702, 66.13%, and 71.31%, respectively, for EC diagnosis. Small interference (si)RNA was employed to silence the CD54 gene in EC cells. A series of assays, including cell counting kit-8, adhesion, wound healing, and Matrigel invasion, were performed to investigate EC viability, adhesive, migratory, and invasive abilities, respectively. The results showed that CD54 promoted EC proliferation, migration, and invasion. Collectively, tissue-derived exosomal proteomics strongly demonstrates that CD54 is a promising biomarker for EC diagnosis and a key molecule for EC development.