Characterization of malignant mesothelioma (MM) from Bap1f/f mouse, which was detected 45 weeks after intrathoracic injection of Adeno-Cre virus. A, Immunoblot confirming loss of Bap1 expression in MM from Bap1 conditional knockout mouse 526, with tumor also showing greatly reduced expression of Nf2 and p16Ink4a as well as Akt activation when compared to normal mesothelial cells (NMC). B, Immunoblots of same lysates as in panel A showing Bap1-related loss of deubiquitination activity in MM 526, as indicated by presence of Ub-H2A in tumor but not in NMC, along with overexpression of product of PRC2 target gene Aldh1a2. In addition to acquired loss of Nf2 expression (panel A), MM 526 shows loss of p-Yap activity, indicative of aberrant Hippo signaling. C, Demonstration that loss of expression of Nf2 and p16Ink4a is acquired, not due to excision of floxed Nf2 and Cdkn2a alleles. Control WT and control Î"/Î" lanes depict sizes of wild type and homozygously floxed (Î"/Î") Bap1, Nf2, and Cdkn2a alleles. DNA from Bap1Î"/Î" mouse tail and matching MM from animal 526 verify that Bap1, but not Nf2 or Cdkn2a, has floxed alleles. Note that the primers used to detect mutant Bap1 allele were designed to bind outside the flox sites, such that a smaller band is seen when Cre recombinase excises Bap1 exon 7.
ARTICLE ABSTRACT
Pleural malignant mesothelioma is a therapy-resistant cancer affecting the serosal lining of the thoracic cavity. Mutations/deletions of BAP1, CDKN2A, and NF2 are the most frequent genetic lesions in human malignant mesothelioma. We introduced various combinations of these deletions in the pleura of conditional knockout (CKO) mice, focusing on the contribution of Bap1 loss. While homozygous CKO of Bap1, Cdkn2a, or Nf2 alone gave rise to few or no malignant mesotheliomas, inactivation of Bap1 cooperated with loss of either Nf2 or Cdkn2a to drive development of malignant mesothelioma in approximately 20% of double-CKO mice, and a high incidence (22/26, 85%) of malignant mesotheliomas was observed in Bap1;Nf2;Cdkn2a (triple)-CKO mice. Malignant mesothelioma onset was rapid in triple-CKO mice, with a median survival of only 12 weeks, and malignant mesotheliomas from these mice were consistently high-grade and invasive. Adenoviral-Cre treatment of normal mesothelial cells from Bap1;Nf2;Cdkn2a CKO mice, but not from mice with knockout of one or any two of these genes, resulted in robust spheroid formation in vitro, suggesting that mesothelial cells from Bap1;Nf2;Cdkn2a mice have stem cell–like potential. RNA-seq analysis of malignant mesotheliomas from triple-CKO mice revealed enrichment of genes transcriptionally regulated by the polycomb repressive complex 2 (PRC2) and others previously implicated in known Bap1-related cellular processes. These data demonstrate that somatic inactivation of Bap1, Nf2, and Cdkn2a results in rapid, aggressive malignant mesotheliomas, and that deletion of Bap1 contributes to tumor development, in part, by loss of PRC2-mediated repression of tumorigenic target genes and by acquisition of stem cell potential, suggesting a potential avenue for therapeutic intervention.
Combinatorial deletions of Bap1, Nf2, and Cdkn2a result in aggressive mesotheliomas, with Bap1 loss contributing to tumorigenesis by circumventing PRC2-mediated repression of oncogenic target genes.