Bap1 Is a Bona Fide Tumor Suppressor: Genetic Evidence from Mouse Models Carrying Heterozygous Germline Bap1 Mutations

Abstract

Individuals harboring inherited heterozygous germline mutations in BAP1 are predisposed to a range of benign and malignant tumor types, including malignant mesothelioma, melanoma, and kidney carcinoma. However, evidence to support a tumor-suppressive role for BAP1 in cancer remains contradictory. To test experimentally whether BAP1 behaves as a tumor suppressor, we monitored spontaneous tumor development in three different mouse models with germline heterozygous mutations in Bap1, including two models in which the knock-in mutations are identical to those reported in human BAP1 cancer syndrome families. We observed spontaneous malignant tumors in 54 of 93 Bap1-mutant mice (58%) versus 4 of 43 (9%) wild-type littermates. All three Bap1-mutant models exhibited a high incidence and similar spectrum of neoplasms, including ovarian sex cord stromal tumors, lung and mammary carcinomas, and spindle cell tumors. Notably, we also observed malignant mesotheliomas in two Bap1-mutant mice, but not in any wild-type animals. We further confirmed that the remaining wild-type Bap1 allele was lost in both spontaneous ovarian tumors and mesotheliomas, resulting in the loss of Bap1 expression. Additional studies revealed that asbestos exposure induced a highly significant increase in the incidence of aggressive mesotheliomas in the two mouse models carrying clinically relevant Bap1 mutations compared with asbestos-exposed wild-type littermates. Collectively, these findings provide genetic evidence that Bap1 is a bona fide tumor suppressor gene and offer key insights into the contribution of carcinogen exposure to enhanced cancer susceptibility. Cancer Res; 76(9); 2836-44. ©2016 AACR.

Figure 1

Schematic diagrams showing cutting sites of the zinc-finger nucleases (ZFNs) and relevant portions of the respective Bap1-mutant alleles, along with genotyping, for Bap1^+/W (A) and Bap1^+/L (B) mutant models.

Figure 2

Collective spectrum of spontaneous tumors observed in all three Bap1-mutant mouse models. Upper pie chart depicts percentages of mice with or without tumors. Lower pie chart indicates percentages of different primary tumors observed. Altogether, neoplastic tumors were identified in 54 of 93 (58%) Bap1-mutant mice. A total of 65 different primary tumors were found in the 54 mice, with 11 mice having two different primary tumors. Mice with bilateral ovarian SCSTs are counted here as one tumor. Percentages of different tumor types refer to proportion of all 65 tumors.

Figure 3

Representative spontaneous ovarian sex cord stromal ovarian tumor (ovarian SCST) from a Bap1^+/W mouse. (A) Macroscopic view of ovarian SCST, normal ovary, fallopian tubes and uterus. (B) H&E staining of same ovarian SCST. (C) Cytoplasmic α-inhibin staining indicating granulosa cell origin of same tumor. (D) IHC for Bap1 showing loss of nuclear staining in most tumor cells. Panels B and D are from serial tissue sections. Original images for panels B–D were at 400× magnification. Representative 50-µm scale bar is shown at lower right in panel B.

Figure 4

Spontaneous MMs seen in a Bap1^+/− mouse (A–C) and a Bap1^+/W mouse (D–F). (A, D) H&E staining showing MM invasion into pancreas and mammary gland, respectively. Abbreviations: MM, malignant mesothelioma; MG, mammary gland; PA, pancreatic acini. Green arrows indicate invasion of pancreatic tissue by MM. IHC of the same tumors showing positive staining for mesothelin (B, E) and nuclear WT1 (C, F). Original images: 200× magnification. Representative 100-µm scale bar is shown in panel D. Note that this is the same tumor as in Fig. 6A,B, which shows invasion of the MM into the pancreatic parenchyma at a lower magnification.

Figure 5

Molecular analysis of Bap1 in spontaneous ovarian SCSTs from several Bap1-mutant mice. (A) aCGH analysis depicting loss of one copy of chromosome 14, including the Bap1 locus (arrow), in 3 ovarian SCSTs shown. Note that copy number loss deviations from the “2-copy” straight line is less obvious in tumors 85 and 34 than in tumor 137, due to mosaicism or the presence of contaminating normal stroma in the former tumors. (B) Immunoblot analysis depicting loss of expression of Bap1 in the same ovarian SCSTs.

Figure 6

Spontaneous and asbestos-induced MM formation in Bap1-mutant mice. (A–C) Bap1 analysis of spontaneous MM from Bap1^+/− knockout mouse, in which tumor cells were isolated by laser-capture microdissection (LCM). H&E staining of tumor prior to (A) and after (B) LCM. Arrow in panel B indicates dissected tumor area. Note that the LCM was performed on the same spontaneous MM shown in Fig. 4A–C and Supplemental Fig. S1; the area chosen for LCM was from a serial section of the same tumor area depicted in Supplemental Fig. 1, which confirmed the diagnosis of MM based on IHC staining and that also revealed loss of nuclear Bap1 staining in many of the tumor cells. Original images presented in panels A and B: 20× magnification. (C) Assessment of Bap1 allelic loss in LCM-isolated tumor cells from the same spontaneous MM see in Bap1^+/− knockout mouse (Left) and from two asbestos-induced MMs from Bap1^+/W mice (Right). Matching tumor and tail DNA were used as templates to amplify a portion of the mouse Bap1 gene in the region encompassing exons 6 and 7 (16). Ethidium bromide gel electrophoresis demonstrates markedly reduced residual wild-type (WT) Bap1 sequences in the spontaneous and asbestos-induced MMs when compared to that of matched tail DNA, indicating that tumor cells have lost the WT Bap1 allele. The Biorad Quantity One program was used to quantitate the intensities of the PCR products. Left, bands corresponding to larger WT Bap1 allele (634 bp) and smaller knockout Bap1 (158 bp) allele, and the ratios of WT to mutant band intensities for each sample are shown below the image. Right, bands representing larger WT Bap1 allele (~300 bp) and smaller knock-in Bap1 “W” allele (~150 bp), the latter being a doublet. Mouse numbers 17 and 128 correspond to asbestos-induced MMs from two different Bap1^+/W knock-in mice. Dotted lines demarcate lanes from different regions of same gel. (D) Kaplan-Meier survival curves showing markedly decreased survival of asbestos-exposed Bap1-mutant knock-in cohorts than in asbestos-exposed WT littermates. Survival differences were highly significant (p < 0.005 for WT vs. Bap1^+/W mice; p <0.008 for WT vs. Bap1^+/L mice). Percentage of deaths due to peritoneal MM was 74% in Bap1^+/W mice and 71% in Bap1^+/L mice compared to 35% of WT animals, which was highly significant (p < 0.005 for WT vs. Bap1^+/W mice; p <0.01 for WT vs. Bap1^+/L mice).