A Mouse Model to Assess STAT3 and STAT5A/B Combined Inhibition in Health and Disease Conditions.

Genetically-engineered mouse models (GEMMs) lacking diseased-associated gene(s) globally or in a tissue-specific manner represent an attractive tool with which to assess the efficacy and toxicity of targeted pharmacological inhibitors. Stat3 and Stat5a/b transcription factors have been implicated in several pathophysiological conditions, and pharmacological inhibition of both transcription factors has been proposed to treat certain diseases, such as malignancies. To model combined inhibition of Stat3 and Stat5a/b we have developed a GEMM harboring a flox Stat3-Stat5a/b allele (Stat5/3loxP/loxP mice) and generated mice lacking Stat3 and Stat5a/b in hepatocytes (Stat5/3Δhep/Δhep). Stat5/3Δhep/Δhep mice exhibited a marked reduction of STAT3, STAT5A and STAT5B proteins in the liver and developed steatosis, a phenotype that resembles mice lacking Stat5a/b in hepatocytes. In addition, embryonic deletion of Stat3 and Stat5a/b (Stat5/3Δ/Δ mice) resulted in lethality, similar to Stat3Δ/Δ mice. This data illustrates that Stat5/3loxP/loxP mice are functional and can be used as a valuable tool to model the combined inhibition of Stat3 and Stat5a/b in tumorigenesis and other diseases.

Afatinib restrains K-RAS-driven lung tumorigenesis.

On the basis of clinical trials using first-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), it became a doctrine that V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-RAS) mutations drive resistance to EGFR inhibition in non-small cell lung cancer (NSCLC). Conversely, we provide evidence that EGFR signaling is engaged in K-RAS-driven lung tumorigenesis in humans and in mice. Specifically, genetic mouse models revealed that deletion of Egfr quenches mutant K-RAS activity and transiently reduces tumor growth. However, EGFR inhibition initiates a rapid resistance mechanism involving non-EGFR ERBB family members. This tumor escape mechanism clarifies the disappointing outcome of first-generation TKIs and suggests high therapeutic potential of pan-ERBB inhibitors. On the basis of various experimental models including genetically engineered mouse models, patient-derived and cell line-derived xenografts, and in vitro experiments, we demonstrate that the U.S. Food and Drug Administration-approved pan-ERBB inhibitor afatinib effectively impairs K-RAS-driven lung tumorigenesis. Our data support reconsidering the use of pan-ERBB inhibition in clinical trials to treat K-RAS-mutated NSCLC.

AKT3 drives adenoid cystic carcinoma development in salivary glands.

Salivary gland cancer is an aggressive and painful cancer, but a rare tumor type accounting for only ~0.5% of cancer cases. Tumors of the salivary gland exhibit heterogeneous histologic and genetic features and they are subdivided into different subtypes, with adenoid cystic carcinomas (ACC) being one of the most abundant. Treatment of ACC patients is afflicted by high recurrence rates, the high potential of the tumors to metastasize, as well as the poor response of ACC to chemotherapy. A prerequisite for the development of targeted therapies is insightful genetic information for driver core cancer pathways. Here, we developed a transgenic mouse model toward establishment of a preclinical model. There is currently no available mouse model for adenoid cystic carcinomas as a rare disease entity to serve as a test system to block salivary gland tumors with targeted therapy. Based on tumor genomic data of ACC patients, a key role for the activation of the PI3K-AKT-mTOR pathway was suggested in tumors of secretory glands. Therefore, we investigated the role of Akt3 expression in tumorigenesis and report that Akt3 overexpression results in ACC of salivary glands with 100% penetrance, while abrogation of transgenic Akt3 expression could revert the phenotype. In summary, our findings validate a novel mouse model to study ACC and highlight the druggable potential of AKT3 in the treatment of salivary gland patients.

Epidermal growth factor signaling protects from cholestatic liver injury and fibrosis.

We have demonstrated that the signal transducer and activator of transcription 3 (STAT3) protects from cholestatic liver injury. Specific ablation of STAT3 in hepatocytes and cholangiocytes (STAT3∆hc) aggravated liver damage and fibrosis in the Mdr2-/- (multidrug resistance 2) mouse model for cholestatic disease. Upregulation of bile acid biosynthesis genes and downregulation of epidermal growth factor receptor (EGFR) expression were observed in STAT3∆hc Mdr2-/- mice but the functional consequences of these processes in cholestatic liver injury remained unclear. Here, we show normal canalicular architecture and bile flow but increased amounts of bile acids in the bile of STAT3∆hc Mdr2-/- mice. Moreover, STAT3-deficient hepatocytes displayed increased sensitivity to bile acid-induced apoptosis in vitro. Since EGFR signaling has been reported to protect hepatocytes from bile acid-induced apoptosis, we generated mice with hepatocyte/cholangiocyte-specific ablation of EGFR (EGFR∆hc) and crossed them to Mdr2-/- mice. Importantly, deletion of EGFR phenocopied deletion of STAT3 and led to aggravated liver damage, liver fibrosis, and hyperproliferation of K19+ cholangiocytes. Our data demonstrate hepatoprotective functions of the STAT3-EGFR signaling axis in cholestatic liver disease. KEY MESSAGE: STAT3 is a negative regulator of bile acid biosynthesis. STAT3 protects from bile acid-induced apoptosis and regulates EGFR expression. EGFR signaling protects from cholestatic liver injury and fibrosis.

Disruption of STAT3 signalling promotes KRAS-induced lung tumorigenesis.

STAT3 is considered to play an oncogenic role in several malignancies including lung cancer; consequently, targeting STAT3 is currently proposed as therapeutic intervention. Here we demonstrate that STAT3 plays an unexpected tumour-suppressive role in KRAS mutant lung adenocarcinoma (AC). Indeed, lung tissue-specific inactivation of Stat3 in mice results in increased Kras(G12D)-driven AC initiation and malignant progression leading to markedly reduced survival. Knockdown of STAT3 in xenografted human AC cells increases tumour growth. Clinically, low STAT3 expression levels correlate with poor survival and advanced malignancy in human lung AC patients with smoking history, which are prone to KRAS mutations. Consistently, KRAS mutant lung tumours exhibit reduced STAT3 levels. Mechanistically, we demonstrate that STAT3 controls NF-κB-induced IL-8 expression by sequestering NF-κB within the cytoplasm, thereby inhibiting IL-8-mediated myeloid tumour infiltration and tumour vascularization and hence tumour progression. These results elucidate a novel STAT3-NF-κB-IL-8 axis in KRAS mutant AC with therapeutic and prognostic relevance.

Modeling cancer using genetically engineered mice.

Genetically engineered mouse (GEM) models have proven to be a powerful tool to study tumorigenesis. The mouse is the preferred complex organism used in cancer studies due to the high number and versatility of genetic tools available for this species. GEM models can mimic point mutations, gene amplifications, short and large deletions, translocations, etc.; thus, most of the genetic aberrations found in human tumors can be modeled in GEM, making GEM models a very attractive system. Furthermore, recent developments in mouse genetics may facilitate the generation of GEM models with increased mutational complexity, therefore resembling human tumors better. Within this review, we will discuss the different possibilities of modeling tumorigenesis using GEM and the future developments within the field.

Growth hormone resistance exacerbates cholestasis-induced murine liver fibrosis.

UNLABELLED: Growth hormone (GH) resistance has been associated with liver cirrhosis in humans but its contribution to the disease remains controversial. In order to elucidate whether GH resistance plays a causal role in the establishment and development of liver fibrosis, or rather represents a major consequence thereof, we challenged mice lacking the GH receptor gene (Ghr(-/-), a model for GH resistance) by crossing them with Mdr2 knockout mice (Mdr2(-/-)), a mouse model of inflammatory cholestasis and liver fibrosis. Ghr(-/-);Mdr2(-/-) mice showed elevated serum markers associated with liver damage and cholestasis, extensive bile duct proliferation, and increased collagen deposition relative to Mdr2(-/-) mice, thus suggesting a more severe liver fibrosis phenotype. Additionally, Ghr(-/-);Mdr2(-/-) mice had a pronounced down-regulation of hepatoprotective genes Hnf6, Egfr, and Igf-1, and significantly increased levels of reactive oxygen species (ROS) and apoptosis in hepatocytes, compared to control mice. Moreover, single knockout mice (Ghr(-/-)) fed with a diet containing 1% cholic acid displayed an increase in hepatocyte ROS production, hepatocyte apoptosis, and bile infarcts compared to their wild-type littermates, indicating that loss of Ghr renders hepatocytes more susceptible to toxic bile acid accumulation. Surprisingly, and despite their severe fibrotic phenotype, Ghr(-/-);Mdr2(-/-) mice displayed a significant decrease in tumor incidence compared to Mdr2(-/-) mice, indicating that loss of Ghr signaling may slow the progression from fibrosis/cirrhosis to cancer in the liver. CONCLUSION: GH resistance dramatically exacerbates liver fibrosis in a mouse model of inflammatory cholestasis, therefore suggesting that GH resistance plays a causal role in the disease and provides a novel target for the development of liver fibrosis treatments.

Heme oxygenase-1 drives metaflammation and insulin resistance in mouse and man.

Obesity and diabetes affect more than half a billion individuals worldwide. Interestingly, the two conditions do not always coincide and the molecular determinants of "healthy" versus "unhealthy" obesity remain ill-defined. Chronic metabolic inflammation (metaflammation) is believed to be pivotal. Here, we tested a hypothesized anti-inflammatory role for heme oxygenase-1 (HO-1) in the development of metabolic disease. Surprisingly, in matched biopsies from "healthy" versus insulin-resistant obese subjects we find HO-1 to be among the strongest positive predictors of metabolic disease in humans. We find that hepatocyte and macrophage conditional HO-1 deletion in mice evokes resistance to diet-induced insulin resistance and inflammation, dramatically reducing secondary disease such as steatosis and liver toxicity. Intriguingly, cellular assays show that HO-1 defines prestimulation thresholds for inflammatory skewing and NF-κB amplification in macrophages and for insulin signaling in hepatocytes. These findings identify HO-1 inhibition as a potential therapeutic strategy for metabolic disease.