Jason D. Heaney

Mice With a Deletion in the Gene for CCAAT/Enhancer-Binding Protein β Are Protected Against Diet-Induced Obesity

The CCAAT/enhancer-binding protein β (C/EBPβ) is required for adipocyte differentiation and maturation. We have studied the role of the transcription factor, C/EBPβ, in the development of diet-induced obesity. Mice with a deletion in the gene for C/EBPβ (C/EBPβ−/−) and wild-type mice were fed a high-fat diet (60% fat) for 12 weeks. The C/EBPβ−/− mice lost body fat, whereas the wild-type mice increased their total body fat on a high-fat diet. The C/EBPβ−/− mice had lower levels of blood triglycerides, free fatty acids, cholesterol, and hepatic triglyceride accumulation compared with the wild-type mice, thus protecting them from diet-induced obesity and fatty liver on a high-fat diet. Deletion of C/EBPβ gene resulted in greatly reducing hepatic lipogenic genes, acetyl CoA carboxylase, and fatty acid synthase and increasing the expression of β-oxidation genes in the brown adipose tissue. CO2 production was significantly higher in the C/EBPβ−/− mice as was the level of uncoupling protein (UCP)-1 and UCP-3 in the muscle. In conclusion, the transcription factor C/EBPβ is an important regulator in controlling lipid metabolism and in the development of diet-induced obesity.

Loss of the Transmembrane but not the Soluble Kit Ligand Isoform Increases Testicular Germ Cell Tumor Susceptibility in Mice

Several genetic variants act as modifiers of testicular germ cell tumor (TGCT) susceptibility in the 129/Sv mouse model of human pediatric TGCTs. One such modifier, the Steel locus, encodes the transmembrane-bound and soluble ligand of the kit receptor. Some (Sl and SlJ) but not all (Sld) mutations of the Steel locus increase TGCT incidence in heterozygous mutant mice. Because Sl and SlJ are large deletions that affect multiple transcripts and Sld is an intragenic deletion of the kit ligand (Kitl) from which only the soluble protein is produced, it was uncertain whether Kitl or a neighboring gene is a modifier of TGCT susceptibility. We tested the effect of the small Steel grizzle-belly (Slgb) deletion on TGCT susceptibility to determine whether Kitl is a TGCT modifier gene. An increase in TGCT incidence was observed in Slgb/+ heterozygotes, and fine mapping of the deletion breakpoints revealed that Kitl is the only conventional gene deleted by the mutation, suggesting that Kitl is the TGCT modifier gene at the Steel locus. Additionally, we propose that soluble KITL in Sld/+ heterozygous mutant mice complements a dosage effect of transmembrane-associated kit ligand on TGCT susceptibility and that the kit receptor (Kit) is haplosufficient for primordial germ cell development.

IL-33 activates tumor stroma to promote intestinal polyposis

Significance Colorectal cancer results from genetic lesions in epithelial cells. However, the tumor microenvironment, which is formed by nonepithelial stromal cells, also plays an important role in this disease. The influence of the microenvironment on tumorigenesis is mediated by paracrine signals between tumor epithelial cells and neighboring stromal cells. We found that expression of interleukin 33 (IL-33), an important mediator of type 2 immunity and wound repair, is induced in epithelial cells of human and mouse intestinal tumors. IL-33 promoted intestinal tumorigenesis in ApcMin/+ mice and activated two stromal cell types, subepithelial myofibroblasts and mast cells, known to mediate intestinal dysplasia. Tumor epithelial cells are proposed to coopt IL-33–mediated immune and wound-healing responses to create a microenvironment favorable to tumorigenesis. Tumor epithelial cells develop within a microenvironment consisting of extracellular matrix, growth factors, and cytokines produced by nonepithelial stromal cells. In response to paracrine signals from tumor epithelia, stromal cells modify the microenvironment to promote tumor growth and metastasis. Here, we identify interleukin 33 (IL-33) as a regulator of tumor stromal cell activation and mediator of intestinal polyposis. In human colorectal cancer, IL-33 expression was induced in the tumor epithelium of adenomas and carcinomas, and expression of the IL-33 receptor, IL1RL1 (also referred to as IL1-R4 or ST2), localized predominantly to the stroma of adenoma and both the stroma and epithelium of carcinoma. Genetic and antibody abrogation of responsiveness to IL-33 in the ApcMin/+ mouse model of intestinal tumorigenesis inhibited proliferation, induced apoptosis, and suppressed angiogenesis in adenomatous polyps, which reduced both tumor number and size. Similar to human adenomas, IL-33 expression localized to tumor epithelial cells and expression of IL1RL1 associated with two stromal cell types, subepithelial myofibroblasts and mast cells, in ApcMin/+ polyps. In vitro, IL-33 stimulation of human subepithelial myofibroblasts induced the expression of extracellular matrix components and growth factors associated with intestinal tumor progression. IL-33 deficiency reduced mast cell accumulation in ApcMin/+ polyps and suppressed the expression of mast cell-derived proteases and cytokines known to promote polyposis. Based on these findings, we propose that IL-33 derived from the tumor epithelium promotes polyposis through the coordinated activation of stromal cells and the formation of a protumorigenic microenvironment.

Phosphoenolpyruvate carboxykinase (Pck1) helps regulate the triglyceride/fatty acid cycle and development of insulin resistance in mice

The aim of this study was to investigate the role of the cytosolic form of phosphoenolpyruvate carboxykinase (Pck1) in the development of insulin resistance. Previous studies have shown that the roles of Pck1 in white adipose tissue (WAT) in glyceroneogenesis and reesterification of free fatty acids (FFA) to generate triglyceride are vital for the prevention of diabetes. We hypothesized that insulin resistance develops when dysregulation of Pck1 occurs in the triglyceride/fatty acid cycle, which regulates lipid synthesis and transport between adipose tissue and the liver. We examined this by analyzing mice with a deletion of the PPARγ binding site in the promoter of Pck1 (PPARE−/−). This mutation reduced the fasting Pck1 mRNA expression in WAT in brown adipose tissue (BAT). To analyze insulin resistance, we performed hyperinsulinemic-euglycemic glucose clamp analyses. PPARE−/− mice were profoundly insulin resistant and had more FFA and glycerol released during the hyperinsulinemic-euglycemic clamp compared with wild-type mice (WT). Finally, we analyzed insulin secretion in isolated islets. We found a 2-fold increase in insulin secretion in the PPARE−/− mice at 16.7 mM glucose. Thus, the PPARE site in the Pck1 promoter is essential for maintenance of lipid metabolism and glucose homeostasis and disease prevention.

Germ cell pluripotency, premature differentiation and susceptibility to testicular teratomas in mice

Testicular teratomas result from anomalies in germ cell development during embryogenesis. In the 129 family of inbred strains of mice, teratomas initiate around embryonic day (E) 13.5 during the same developmental period in which female germ cells initiate meiosis and male germ cells enter mitotic arrest. Here, we report that three germ cell developmental abnormalities, namely continued proliferation, retention of pluripotency, and premature induction of differentiation, associate with teratoma susceptibility. Using mouse strains with low versus high teratoma incidence (129 versus 129-Chr19MOLF/Ei), and resistant to teratoma formation (FVB), we found that germ cell proliferation and expression of the pluripotency factor Nanog at a specific time point, E15.5, were directly related with increased tumor risk. Additionally, we discovered that genes expressed in pre-meiotic embryonic female and adult male germ cells, including cyclin D1 (Ccnd1) and stimulated by retinoic acid 8 (Stra8), were prematurely expressed in teratoma-susceptible germ cells and, in rare instances, induced entry into meiosis. As with Nanog, expression of differentiation-associated factors at a specific time point, E15.5, increased with tumor risk. Furthermore, Nanog and Ccnd1, genes with known roles in testicular cancer risk and tumorigenesis, respectively, were co-expressed in teratoma-susceptible germ cells and tumor stem cells, suggesting that retention of pluripotency and premature germ cell differentiation both contribute to tumorigenesis. Importantly, Stra8-deficient mice had an 88% decrease in teratoma incidence, providing direct evidence that premature initiation of the meiotic program contributes to tumorigenesis. These results show that deregulation of the mitotic-meiotic switch in XY germ cells contributes to teratoma initiation.

Deletion of eIF2beta suppresses testicular cancer incidence and causes recessive lethality in agouti-yellow mice

The agouti-yellow (A(y)) deletion is the only genetic modifier known to suppress testicular germ cell tumor (TGCT) susceptibility in mice or humans. The A(y) mutation deletes Raly and Eif2s2, and induces the ectopic expression of agouti, all of which are potential TGCT-modifying mutations. Here we report that the reduced TGCT incidence of heterozygous A(y) males and the recessive embryonic lethality of A(y) are caused by the deletion of Eif2s2, the beta subunit of translation initiation factor eIF2. We found that the incidence of affected males was reduced 2-fold in mice that were partially deficient for Eif2s2 and that embryonic lethality occurred near the time of implantation in mice that were fully deficient for Eif2s2. In contrast, neither reduced expression of Raly in gene-trap mice nor ectopic expression of agouti in transgenic or viable-yellow (A(vy)) mutants affected TGCT incidence or embryonic viability. In addition, we provide evidence that partial deficiency of Eif2s2 attenuated germ cell proliferation and differentiation, both of which are important to TGCT formation. These results show that germ cell development and TGCT pathogenesis are sensitive to the availability of the eIF2 translation initiation complex and to changes in the rate of translation.

Transgenerational epigenetic effects of the Apobec1 cytidine deaminase deficiency on testicular germ cell tumor susceptibility and embryonic viability

Environmental agents and genetic variants can induce heritable epigenetic changes that affect phenotypic variation and disease risk in many species. These transgenerational effects challenge conventional understanding about the modes and mechanisms of inheritance, but their molecular basis is poorly understood. The Deadend1 (Dnd1) gene enhances susceptibility to testicular germ cell tumors (TGCTs) in mice, in part by interacting epigenetically with other TGCT modifier genes in previous generations. Sequence homology to A1cf, the RNA-binding subunit of the ApoB editing complex, raises the possibility that the function of Dnd1 is related to Apobec1 activity as a cytidine deaminase. We conducted a series of experiments with a genetically engineered deficiency of Apobec1 on the TGCT-susceptible 129/Sv inbred background to determine whether dosage of Apobec1 modifies susceptibility, either alone or in combination with Dnd1, and either in a conventional or a transgenerational manner. In the paternal germ-lineage, Apobec1 deficiency significantly increased susceptibility among heterozygous but not wild-type male offspring, without subsequent transgenerational effects, showing that increased TGCT risk resulting from partial loss of Apobec1 function is inherited in a conventional manner. By contrast, partial deficiency in the maternal germ-lineage led to suppression of TGCTs in both partially and fully deficient males and significantly reduced TGCT risk in a transgenerational manner among wild-type offspring. These heritable epigenetic changes persisted for multiple generations and were fully reversed after consecutive crosses through the alternative germ-lineage. These results suggest that Apobec1 plays a central role in controlling TGCT susceptibility in both a conventional and a transgenerational manner.

Testicular Germ Cell Tumors in Mice

Testicular germ cell tumors (TGCTs) are the most common cancer affecting young men. Although TGCTs are common and the genetic component of susceptibility is unusually strong, discovery of TGCT susceptibility genes in humans has been challenging. The 129/Sv inbred mouse strain is an important experimental model for studying the genetic control of TGCT susceptibility. It is the only inbred mouse strain with an appreciable frequency of spontaneous TGCTs. TGCTs in 129/Sv males share various developmental and histological characteristics with human pediatric TGCTs. As in humans, susceptibility in 129/Sv is a genetically complex trait that is too complex for conventional genetic approaches. However, several genetic variants, when congenic or isogenic on the 129/Sv background, act as genetic modifiers of TGCT susceptibility. Alternative experimental approaches based on these modifier genes can be used to unravel the complex genetic control of TGCT susceptibility. We discuss the application of modifier genes in genetic interaction tests and sensitized polygenic trait analyses toward the understanding of the complex genetics and biology of TGCT susceptibility in mice.

Deficiency of Splicing Factor 1 Suppresses the Occurrence of Testicular Germ Cell Tumors

Testicular germ cell tumors (TGCT) originate from germ cells. The 129-Ter and M19 (129.MOLF-Chr19 consomic) mouse strains have extremely high incidences of TGCTs. We found that the expression levels of Sf1-encoded splicing factor 1 (SF1) can modulate the incidence of TGCTs. We generated mice with inactivated Sf1. Sf1 null mice (Sf1-/-) died before birth. Mice with one intact allele of Sf1 (Sf1+/-) were viable but expressed reduced levels of Sf1. When Sf1-deficient mice (Sf1+/-) were crossed to the 129-Ter and M19 strains, we observed decreased incidence of TGCTs in Sf1+/-;Ter and Sf1+/-;M19/+ mice compared with that in control cohorts. Therefore, Sf1 deficiency protects against TGCT development in both strains. Sf1 is expressed in the testes. We found that Sf1 levels vary significantly in the testes of inbred strains such as 129 and MOLF, and as such Sf1 is an oncogenic tumor-susceptibility factor from 129. Our results also highlight the complications involved in evaluating Sf1 levels and TGCT incidences. When a large number of tumor-promoting factors are present in a strain, the protective effect of lower Sf1 levels is masked. However, when the dosage of tumor-promoting factors is reduced, the protective effect of lower Sf1 levels becomes apparent. SF1 is involved in splicing of specific pre-mRNAs in cells. Alternate splicing generates the complex proteosome in eukaryotic cells. Our data indicate that Sf1 levels in mouse strains correlate with their incidences of TGCTs and implicate the importance of splicing mechanisms in germ cell tumorigenesis.

High-Fat Diet-Induced Complement Activation Mediates Intestinal Inflammation and Neoplasia, Independent of Obesity

Obesity and related metabolic disturbances are closely associated with pathologies that represent a significant burden to global health. Epidemiological and molecular evidence links obesity and metabolic status with inflammation and increased risk of cancer. Here, using a mouse model of intestinal neoplasia and strains that are susceptible or resistant to diet-induced obesity, it is demonstrated that high-fat diet-induced inflammation, rather than obesity or metabolic status, is associated with increased intestinal neoplasia. The complement fragment C5a acts as the trigger for inflammation and intestinal tumorigenesis. High-fat diet induces complement activation and generation of C5a, which in turn induces the production of proinflammatory cytokines and expression of proto-oncogenes. Pharmacological and genetic targeting of the C5a receptor reduced both inflammation and intestinal polyposis, suggesting the use of complement inhibitors for preventing diet-induced neoplasia. Implications: This study characterizes the relations between diet and metabolic conditions on risk for a common cancer and identifies complement activation as a novel target for cancer prevention. Mol Cancer Res; 14(10); 953–65. ©2016 AACR.