Determination of the Potential Tumor-Suppressive Effects of Gsdme in a Chemically Induced and in a Genetically Modified Intestinal Cancer Mouse Model

cancers Article Determination of the Potential Tumor-Suppressive Effects of Gsdme in a Chemically Induced and in a Genetically Modified Intestinal Cancer Mouse Model Lieselot Croes 1,2 , Erik Fransen 1,3 , Marieke Hylebos 1,2 , Kimberly Buys 1,2 , Christophe Hermans 2 , Glenn Broeckx 4 , Marc Peeters 2,5 , Patrick Pauwels 2,4 , Ken Op de Beeck 1,2 and Guy Van Camp 1,2, * 1 2 3 4 5 * Center of Medical Genetics, University of Antwerp, Prins Boudewijnlaan 43/6, Edegem, BE-2650 Antwerp, Belgium Center for Oncological Research, University of Antwerp, Universiteitsplein 1, Wilrijk, BE-2610 Antwerp, Belgium StatUa Center for Statistics, University of Antwerp, Prinsstraat 13, BE-2000 Antwerp, Belgium Department of Pathology, Antwerp University Hospital, Wilrijkstraat 10, Edegem, BE-2650 Antwerp, Belgium Department of Medical Oncology, Antwerp University Hospital, Wilrijkstraat 10, Edegem, BE-2650 Antwerp, Belgium Correspondence: ; Tel.: +32-03-275-9762 Received: 11 July 2019; Accepted: 13 August 2019; Published: 20 August 2019



 Abstract: Gasdermin E (GSDME), also known as deafness autosomal dominant 5 (DFNA5) and previously identified to be an inducer of regulated cell death, is frequently epigenetically inactivated in different cancer types, suggesting that GSDME is a tumor suppressor gene. In this study, we aimed to evaluate the tumor-suppressive effects of GSDME in two intestinal cancer mouse models. To mimic the silencing of GSDME by methylation as observed in human cancers, a Gsdme knockout (KO) mouse was developed. The effect of GSDME on tumorigenesis was studied both in a chemically induced and in a genetic intestinal cancer mouse model, as strong evidence shows that GSDME plays a role in human colorectal cancer and representative mouse models for intestinal cancer are available. Azoxymethane (AOM) was used to induce colorectal tumors in the chemically induced intestinal cancer model (n = 100). For the genetic intestinal cancer model, Apc1638N/+ mice were used (n = 37). In both experiments, the number of mice bearing microscopic proliferative lesions, the number and type of lesions per mouse and the histopathological features of the adenocarcinomas were compared between Gsdme KO and wild type (WT) mice. Unfortunately, we found no major differences between Gsdme KO and WT mice, neither for the number of affected mice nor for the multiplicity of proliferative lesions in the mice. However, recent breakthroughs on gasdermin function indicate that GSDME is an executioner of necrotic cell death. Therefore, it is possible that GSDME may be important for creating an inflammatory microenvironment around the tumor. This is in line with the trend towards more severe inflammation in WT compared to Gsdme KO mice, that we observed in our study. We conclude that the effect of GSDME in tumor biology is probably more subtle than previously thought. Keywords: Gsdme; Dfna5; knockout; mouse model; colorectal cancer; intestinal cancer; Apc; AOM; azoxymethane; inflammation Cancers 2019, 11, 1214; doi:10.3390/cancers11081214 www.mdpi.com/journal/cancers Cancers 2019, 11, 1214 2 of 20 1. Introduction Gasdermin E (GSDME), also known as deafness autosomal dominant 5 (DFNA5), was discovered in our lab as a gene responsible for a specific form of nonsyndromic, autosomal dominant hearing loss [1]. Previously, we demonstrated that GSDME has the capacity to induce regulated cell death [2–4]. Since its discovery in 1998, a number of studies on GSDME have been published, pointing towards a possible involvement in cancer [2–17]. Moreover, genomic methylation screens unveiled GSDME as a possible tumor suppressor gene [11,13,14]. Furthermore, epigenetic silencing through GSDME methylation was previously shown in primary gastric [14], breast [6,7,12], and colorectal cancer [5,9,13]. In addition, GSDME expression was significantly downregulated, both in colon cancer samples and in colorectal cancer cell lines [13]. Finally, in vitro studies showed an increase in cellular invasiveness, colony numbers, colony size, and cell growth in colorectal cancer cell lines after GSDME knock-down [13]. Forced expression of GSDME, on the other hand, decreased cell growth and colony forming ability. In conclusion, these data suggested that GSDME is a tumor suppressor gene, which is often epigenetically inactivated through DNA methylation in different types of cancer. In this study, we aimed to determine the potential tumor-suppressive effects of Gsdme both in a chemically induced and in a genetically modified intestinal cancer mouse model, given the strong evidence that GSDME plays a role in human colorectal cancer [5,9,13] and good, representative mouse models for intestinal cancer are available [18–26]. To mimic the silencing of GSDME by methylation, as observed in human cancers, a Gsdme knockout (KO) mouse model was developed. For the chemically induced intestinal cancer model, azoxymethane (AOM) was used. AOM is a chemical agent that can initiate cancer by alkylation of DNA, thereby facilitating base mismatch [19]. The AOM model recapitulates many of the histopathological features associated with the multistage progression of human sporadic colorectal cancers [19,27]. Moreover, it has already been successfully used in numerous studies investigating factors that play a role in the modulation of tumor initiation and progression [28–30]. The model that was used in this study, with repeated intraperitoneal (i.p.) injections, is especially useful for studying factors that drive spontaneous tumor progression [19]. For the genetic intestinal cancer model, Apc1638N/+ mice were used. Mutations in the APC gene are found in the earliest stages of the adenoma-carcinoma pathway and therefore play a crucial role in tumor formation and progression. The Apc1638N/+ mouse model was chosen because it is a well-documented strain of genetically engineered Apc mice with a C57BL/6 background [20,21]. Compared to the frequently used ApcMin/+ mice, Apc1638N/+ mice have an attenuated intestinal phenotype with fewer tumors, occurring at a later time, which can progress into adenocarcinomas [20,21]. Therefore, Apc1638N/+ mice are suitable for determining the effects of additional factors, such as Gsdme, in carcinogenesis. Apc1638N/+ mice are known to progressively develop aberrant crypt foci, colonic polyps, and tumors of the small intestine, both benign adenomas and malignant adenocarcinomas, in the duodenum and jejunum [21,31]. In this study, we compared the number of mice bearing microscopic proliferative lesions, the number and type of lesions per mouse and the histopathological features of the adenocarcinomas between Gsdme KO and wild type (WT) mice. 2. Results 2.1. Validation of the Gsdme KO Mouse Model To confirm the Gsdme KO status of the generated mice, we performed Gsdme mRNA and protein expression analyses. Gsdme mRNA expression analyses on Gsdme KO (n = 7) and WT (n = 9) mice were performed, both on brai (...truncated)