Whole exome sequencing of an asbestos-induced wild-type murine model of malignant mesothelioma

Our plain-English summary of Whole exome sequencing of an asbestos-induced wild-type murine model of malignant mesothelioma, by S. Sneddon, A.M. Patch, I.M. Dick, et al (BMC Cancer, 2017).

The study

• Authors: S. Sneddon, A.M. Patch, I.M. Dick, S. Kazakoff, J.V. Pearson, N. Waddell, R.J.N. Allcock, R.A. Holt, B.W.S. Robinson, J. Creaney
• Journal: BMC Cancer
• Published: 2017;17(1):396
• Cited by: 26 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Malignant mesothelioma (MM) is an aggressive cancer of the pleural and peritoneal cavities caused by exposure to asbestos. Asbestos-induced mesotheliomas in wild-type mice have been used extensively as a preclinical model because they are phenotypically identical to their human counterpart. However, it is not known if the genetic lesions in these mice tumours are similar to in the human disease, a prerequisite for any new preclinical studies that target genetic abnormalities.

Methods: We performed whole exome sequencing of fifteen asbestos-induced murine MM tumour cell lines from BALB/c, CBA and C57BL/6 mouse strains and compared the somatic mutations and copy number variations with those recurrently reported in human MM. We then catalogued and characterised the mutational landscape of the wild-type murine MM tumours. Quantitative RT-PCR was used to interrogate the expression of key MM genes of interest in the mRNA.

Results: Consistent with human MM tumours, we identified homozygous loss of the tumour suppressor Cdkn2a in 14/15 tumours. One tumour retained the first exon of both of the p16INK4a and p19ARF isoforms though this tumour also contained genetic amplification of Myc resulting in increased expression of the c-Myc proto-oncogene in the mRNA. There were no chromosomal losses in either the Bap1 or Nf2 regions. One tumour harbored homozygous loss of Trp53 in the DNA. Mutation rates were similar in tumours generated in the CBA and C57BL/6 strains when compared to human MM. Interestingly, all BALB/c tumour lines displayed high mutational loads, consistent with the known mutator phenotype of the host strain. The Wnt, MAPK and Jak-STAT signaling pathways were found to be the most commonly affected biological pathways. Mutations and copy number deletions also occurred in the Hedgehog and Hippo pathways.

Conclusions: These data suggest that in the wild-type murine model asbestos causes mesotheliomas in a similar way to in human MM. This further supports the notion that the murine model of MM represents a genuine homologue of the human disease, something uncommon in cancer, and is thus a valuable tool to provide insight into MM tumour development and to aide the search for novel therapeutic strategies.

In plain English

This was a laboratory study in mice. The authors sequenced the protein-coding DNA (exome) of 15 mesothelioma cell lines. The lines had been grown from abdominal fluid of BALB/c (4), CBA (5) and C57BL/6 (6) mice given crocidolite asbestos injections into the peritoneum. The authors compared the changes with those reported in human mesothelioma. Both copies of the region containing the tumour suppressor gene Cdkn2a were deleted in all 15 lines, although one line (AE17) kept the first exon of both forms of the gene, and Cdkn2a messenger RNA was not detected in any sample. A region containing the Myc gene was amplified in 9 of 15 lines. No mutations were found in Bap1, Nf2 or Lats2, though 3 of 15 lines had Lats2 deletions. One line had lost both copies of Trp53. The median mutation rate was 0.7 per million bases (3.9 in BALB/c lines). The authors concluded that asbestos causes mesothelioma in these mice through Cdkn2a loss. They noted that the mouse tumours were peritoneal, whereas most human tumours are pleural, and that cell culture may explain some changes.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory animal study: whole exome sequencing and gene expression testing (quantitative RT-PCR) of asbestos-induced mouse mesothelioma cell lines, compared with genetic changes reported in human mesothelioma
• Who was studied: 15 mouse mesothelioma cell lines established between 1992 and 2005 from BALB/c (n = 4), CBA (n = 5) and C57BL/6 (n = 6) mice, with control DNA and RNA from current-generation mice of each strain (Animal Resource Centre, Murdoch, Western Australia)
• Exposure: Crocidolite asbestos injected into the peritoneal (abdominal) cavity of the mice
• Main results:
  • Both copies of a region containing the tumour suppressor gene Cdkn2a were deleted in all 15 mouse cell lines. This was the most significant copy-number change found.
  • In one cell line (AE17), exon 1 of both Cdkn2a isoforms was retained. Quantitative real-time PCR found no Cdkn2a expression in messenger RNA in any sample.
  • A region containing the Myc oncogene was amplified in 9 of 15 cell lines. Myc expression was increased 150-fold in AE17.
  • No mutations were found in Bap1, Nf2 or Lats2, the genes most commonly mutated in human mesothelioma. Copy-number deletions affected Lats2 in 3 of 15 lines and Setd2 in 2 of 15 lines, without reduced messenger RNA expression.
  • One cell line (AC29), from a CBA mouse, had a deletion of both copies of Trp53.
  • Across the 15 samples, 1286 somatic single-base changes and small insertions or deletions were found in 1136 genes.
  • The median mutation rate was 0.7 mutations per million bases overall. By strain, it was 3.9 for BALB/c, 0.6 for CBA and 0.7 for C57BL/6 lines.
  • Three biological pathways were enriched among the significantly altered genes: regulation of autophagy, RIG-I-like receptor signaling and Jak-STAT signaling. In addition, 5 of 15 samples had missense mutations in Wnt signaling pathway genes.
• The authors' conclusion: The authors concluded that asbestos exposure in wild-type mice causes mesothelioma through homozygous loss of Cdkn2a, which they described as an initiating event. They found a mutation rate similar to human mesothelioma. They considered the wild-type mouse model a tool for studying the molecular basis of human mesothelioma.
• Limitations the authors state:
  • The mouse tumours developed in the peritoneum, whereas most human mesotheliomas develop in the pleura.
  • Selection pressure from cell culture conditions may account for some copy-number changes. The authors noted that the number of changes did not correlate with time in culture.
  • The study was too small (underpowered) to perform a full mutational signature analysis.
  • Because post-transcriptional modification could account for loss of other tumour suppressors, the authors said they could not conclusively say that Cdkn2a deletion is an independent driver of mesothelioma.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Lung cancer, Mesothelioma.

Medical terms in this study, explained in our Lexicon: Malignant Mesothelioma, Mesothelioma, Somatic Mutation, CDKN2A, BAP1.

Related studies

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• Whole-exome sequencing reveals frequent genetic alterations in BAP1, NF2, CDKN2A, and CUL1 in malignant pleural mesothelioma — G. Guo, J. Chmielecki, et al. (2015)
• Whole exome and targeted deep sequencing identify genome-wide allelic loss and frequent SETDB1 mutations in malignant pleural mesotheliomas — H.C. Kang, H.K. Kim, et al. (2016)
• High levels of MDM2 are not correlated with the presence of wild-type p53 in human malignant mesothelioma cell lines — S. Ungar, A. Van de Meeren, et al. (1996)
• Germline and somatic variant identification using BGISEQ-500 and HiSeq X Ten whole genome sequencing — A.M. Patch, K. Nones, et al. (2018)
• Clinical Implications of Real-time Integrative Sequencing in Management of Patients With Suspected Germline BAP1 Mutations — S. Sengupta, A.C. Weyand, et al. (2019)

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