Gasdermin-B Pro-Tumor Function in Novel Knock-in Mouse Models Depends on the in vivo Biological Context

Abstract

Gasdermins (GSDM) genes play complex roles in inflammatory diseases and cancer. Gasdermin-B (GSDMB) is frequently upregulated in human cancers, especially in HER2-amplified breast carcinomas, and can promote diverse pro-tumor functions (invasion, metastasis, therapy-resistance). In particular, the GSDMB shortest translated variant (isoform 2; GSDMB2) increases aggressive behavior in breast cancer cells. Paradoxically, GSDMB can also have tumor suppressor (cell death induction) effects in specific biological contexts. However, whether GSDMB has inherent oncogenic, or tumor suppressor function in vivo has not been demonstrated yet in preclinical mouse models, since mice lack GSDMB orthologue. Therefore, to decipher GSDMB cancer functions in vivo we first generated a novel knock-in mouse model (R26-GB2) ubiquitously expressing human GSDMB2. The comprehensive histopathological analysis of multiple tissues from 75 animals showed that nucleus-cytoplasmic GSDMB2 expression did not clearly affect the overall frequency nor the histology of spontaneous neoplasias (mostly lung carcinomas), but associated with reduced incidence of gastric tumors, compared to wildtype animals. Next, to assess specifically the GSDMB2 roles in breast cancer, we generated two additional double transgenic mouse models, that co-express GSDMB2 with either the HER2/NEU oncogene (R26-GB2/MMTV-NEU mice) or the Polyoma middle-T antigen (R26-GB2/MMTV-PyMT) in breast tumors. Consistent with the pro-tumor effect of GSDMB in HER2+ human breast carcinomas, R26-GB2/MMTV-NEU GSDMB2-positive mice have double breast cancer incidence than wildtype animals. By contrast, in the R26-GB2/MMTV-PyMT model of fast growing and highly metastatic mammary tumors, GSDMB2 expression did not significantly influence cancer development nor metastatic potential. In conclusion, our data prove that GSDMB2 in vivo pro-tumor effect is evidenced only in specific biological contexts (in concert with the HER2 oncogene), while GSDMB2 alone does not have overall intrinsic oncogenic potential in genetically modified mice. Our novel models are useful to identify the precise stimuli and molecular mechanisms governing GSDMB functions in neoplasias and can be the basis for the future development of additional tissue-specific and context-dependent cancer models.

Keywords: Cancer Progression; Gasdermins; novel mouse models; pyroptosis; tumorigenesis.

FIGURE 1

Generation of knock-in mouse models harboring human GSDMB isoform 2 transcript within the ROSA26 locus. (A) Schematic representation of the GSDMB isoform 2 (GB2) targeted floxed allele (top, conditional model) and the expression allele (bottom) within the ROSA26 (R26) locus. The construct contains a splicing acceptor signal (SA), the PGK-neomycin-STOP cassette flanked by LoxP sites, the human GSDMB2 isoform 2 cDNA sequence (GB2) fused with HA tag, followed by the IRES-GFP reporter gene. After crossing with EIIa-Cre strain (red arrow), the Cre-mediated excision of the PGK-neomycin-stop element allows the ubiquitous expression of the GB2-HA/GFP tandem under the control of the ROSA26 promoter (R26-GB2). The primer pairs for PCR analyses are also detailed (gray arrows). (B) Diagnostic PCR analysis of positive ES cell clones showing the proper insertion of the recombinant R26-STOP-GB2 allele. H20, Negative control. (C) Examples of genotyping PCR analysis (primers in gray), demonstrating the excision of neo-stop cassete in Cre+/GB2 mice. (D) Ubiquitous expression of the transgenes is verified by GFP fluorescent emission of fresh tail skin (top) and testes (bottom) from WT and GSDMB-positive R26-GB2 mice. Full-length gels are presented in Supplementary Figure S1.

FIGURE 2

Ubiquitous expression of GSDMB2-HA and GFP in the R26-GB2 mouse model. (A) Representative western blot analysis in different tissues from GB2 (+/- heterozygous; +/+ homozygous) and WT (control) littermate mice. GAPDH was used as a loading control. C+, MCF7 exogenously expressing GSDMB2-HA and GFP genes were used as a positive control. (B–C) Expression of GSDMB2-HA and GFP transgenes by WB (B) and GFP by flow cytometry (C) in whole blood leukocytes from R26-GB2 mice. Full-length blots are presented in Supplementary Figure S1.

FIGURE 3

Immunohistochemical expression of GSDMB2-HA in different tissues of the R26-GB2 mouse model. Representative images of tissues from homozygous (GB2+/+) and control (WT) mouse littermates. * Unspecific staining. Scale bar, 100 µm.

FIGURE 4

Immunohistochemical expression of GSDMB2-HA in spontaneous lung carcinomas from the R26-GB2 mouse model. Representative images of lung cancers from homozygous (GB2+/+), heterozygous (GB2+/-) and control (WT) mice. Note the stronger expression of GSDMB2-HA in GB2+/+ than GB2+/- cancer cells and the negative staining in the WT condition. N: normal lung bronchiole. Scale bar, 100 µm.

FIGURE 5

GSDMB2-HA expression in primary breast carcinomas and corresponding lung metastasis from R26-GB2/MMTV-NEU model and R26-GB2/MMTV-PyMT mice. (A) Representative images of the GSDMB2-HA and HER2 immunohistochemical expression in primary tumors and metastatic foci from WT, GB2+/- and GB2+/+ GB2/MMTV-NEU mice of 15 months age. (B) Representative images of the GSDMB2-HA staining from GB2+/+ and WT GB2/MMTV-NEU mice of 15 weeks age. Scale bar, 100 µm. (C) Comparison of GSDMB2-HA expression between tumors from the two models by Western blot. GAPDH was used as loading control.

FIGURE 6

Effect of GSDMB2 on breast cancer generation and cancer progression in the R26-GB2/MMTV-NEU and R26-GB2/MMTV-PyMT mouse models. (A) Comparisons between GB2+ (GB2+/+ and GB2+/-combined) and WT mice of the GB2/MMTV-NEU model (median age 15 months). All mice were heterozygous for HER2/NEU oncogene. (B) Comparisons between GB2+/+ and WT mice of the GB2/MMTV-PyMT model (15 weeks of age). All mice were heterozygous for PyMT oncogene. Tumor latency: time until detection of palpable mammary tumors. Total tumor weight: Each data point represents the added weight of all tumors for each mouse. Proliferation rate: Percentage of PCNA-positive staining (only the biggest tumor of each mouse). Lung metastasis foci: number of metastatic lesions (only animals with metastasis). Graphs show all data points, mean values (line) and standard deviations (error bars). Statistical differences were tested by Student’s t-test.