In Vivo Analysis of the Role of Gasdermin-B (Gsdmb) in Cancer Using Novel Knock-in Mouse Models

Abstract

Background: Gasdermin-B gene (GSDMB) is frequently over-expressed in tumors, and its shortest translated variant (isoform 2; GSDMB2) increases aggressive behavior in breast cancer cells. Paradoxically, GSDMB could have either pro-tumor or tumor suppressor properties depending on the biological context. Since GSDMB gene is not present in the mouse genome, deciphering fully the functional roles of GSDMB in cancer requires novel in vivo models. Methods: We first generated by gene targeting a conditional knock-in mouse model (R26-STOP-GB2) harboring human GSDMB2 transcript within the ROSA26 locus. We next derived the R26-GB2 model ubiquitously expressing GSDMB2 in multiple tissues (confirmed by western blot and immunohistochemistry) and performed a comprehensive histopathological analysis in multiple tissues from 75 male and female mice up to 18 months of age. Additionally, we produced the double transgenic model R26-GB2/MMTV-PyMT, co-expressing GSDMB2 and the Polyoma-Middle-T oncogene, and assessed breast cancer generation and progression in GSDMB2-homozygous (n=10) and control (n=17) female mice up to 15 weeks of age. Results: In the R26-GB2 model, which showed different GSDMB2 cytoplasmic and/or nuclear localization among tissues, we investigated if GSDMB2 expression had intrinsic tumorigenic activity. 41% of mice developed spontaneous lung tumors, but neither the frequency nor the histology of these neoplasias was significantly different from wildtype animals. Strikingly, while 17% control mice developed gastric carcinomas, no GSDMB2-positive mice did. No other tumor types or additional histological alterations were frequently seen in these mice. In the R26-GB2/MMTV-PyMT model, the strong nucleus-cytoplasmic GSDMB2 expression in breast cancer cells did not significantly affect cancer formation (number of tumors, latency, tumor weight, histology or proliferation) or lung metastasis potential compared to controls. Conclusions: GSDMB2 expression alone does not have an overall tumorigenic potential in mice, but it might reduce gastric carcinogenesis. Contrary to human cancers, GSDMB2 upregulation does not significantly affect breast cancer generation and progression in mouse models. However, to evidence the GSDMB functions in cancer and other pathologies in vivo may require the presence of specific stimulus or cellular contexts. Our novel mouse strains will serve as the basis for the future development of more precise tissue-specific and context-dependent cancer models.

Distinct GSDMB protein isoforms and protease cleavage processes differentially control pyroptotic cell death and mitochondrial damage in cancer cells

“…These results are in line with the enhanced in vivo aggressiveness and tumorigenic potential of GSDMB2 observed in breast cancer xenografts (47) and GSDMB2/HER2 knock-in mouse models (40).…”

“…We proved that expression of GSDMB1-2 would be beneficial for tumor cells, as these variants are resistant to pyroptosis-activation mediated by GZMA after immune attack. Indeed, the shortest variant (GSDMB2 which lacks exons 6-7) is particularly advantageous for breast cancer cells, as it increases in vivo tumor development and metastasis in MCF7 cell xenografts in immunodeficient mice and enhances tumorigenesis in an immune-proficient Knockin model of GSDMB2/HER2 breast cancer (40). Consistent with this, GSDMB2 mRNA is the most upregulated transcript in the TCGA breast cancer cohort (63) significantly associates with unfavorable clinical-pathological and prognostic markers.…”

“…Based our results, we reasoned that the differential expression of GSDMB variants can have an impact on the biological and clinical behavior of GSDMB-positive tumors, since those mostly expressing exon 6-null isoforms (GSDMB1-2) should not have GSDMB-mediated pyroptotic anti-tumor function. To test this hypothesis, we focus on breast tumors, since we have previously demonstrated that in mammary carcinomas, GSDMB over-expression has prognostic value and promotes multiple pro-tumor effects in vitro and in vivo (39)(40)(41)47). First, in 1 093 breast cancer patients from The Cancer Genome Atlas (TCGA) (63) we observed that GSDMB2 mean expression was generally higher than the other isoforms (Figure 6A).…”

“…In tumors, GSDMB promotes either pro-tumor or anti-tumor functions depending on the biological context (37). GSDMB is frequently co-amplified with HER2/Erbb2 oncogene in breast carcinomas (39), where GSDMB over-expression promotes tumorigenesis (40), invasion, metastasis, and resistance to therapy (39,41). Paradoxically, GSDMB can also have an anti-tumour role if its pore-forming pyroptotic function is activated in cancer cells either with a GSDMB-targeted nanotherapy (41) or extrinsically through its cleavage by GZMA (17).…”