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The surface of intestinal epithelial cells is covered by the brush border, which consists of densely packed cellular extrusions called microvilli. Until recently, microvilli have not been known to be interconnected. In 2014, a protein complex, called the intermicrovillar adhesion complex (IMAC) which is located at the tips of the microvilli and responsible for the regular spatial organization of the brush border, was identified. Deletion of IMAC components such as cadherin-related family member-2 (CDHR2) in mice resulted in microvillus disorganization and fanning, a structural aberration that is also found in the brush border of patients with inflammatory bowel disease. The etiology of inflammatory bowel disease has been primarily associated with dysfunctional mucosal immunity, but the discovery of the IMAC may encourage theories of an epithelial origin. Here, possible effects of the brush border on the gut barrier function and intestinal inflammation are discussed proposing that the IMAC protects against inflammation through its microvillus cross-linking function.

Janus kinase Tyk2 is implicated in cancer immune surveillance, but its role in solid tumors is not well defined. We used Tyk2 knockout mice (Tyk2Δ/Δ) and mice with conditional deletion of Tyk2 in hematopoietic (Tyk2ΔHem) or intestinal epithelial cells (Tyk2ΔIEC) to assess their cell type-specific functions in chemically induced colorectal cancer. All Tyk2-deficient mouse models showed a higher tumor burden after AOM-DSS treatment compared to their corresponding wild-type controls (Tyk2+/+ and Tyk2fl/fl), demonstrating tumor-suppressive functions of Tyk2 in immune cells and epithelial cancer cells. However, specific deletion of Tyk2 in hematopoietic cells or in intestinal epithelial cells was insufficient to accelerate tumor progression, while deletion in both compartments promoted carcinoma formation. RNA-seq and proteomics revealed that tumors of Tyk2Δ/Δ and Tyk2ΔIEC mice were immunoedited in different ways with downregulated and upregulated IFNγ signatures, respectively. Accordingly, the IFNγ-regulated immune checkpoint Ido1 was downregulated in Tyk2Δ/Δ and upregulated in Tyk2ΔIEC tumors, although both showed reduced CD8+ T cell infiltration. These data suggest that Tyk2Δ/Δ tumors are Ido1-independent and poorly immunoedited while Tyk2ΔIEC tumors require Ido1 for immune evasion. Our study shows that Tyk2 prevents Ido1 expression in CRC cells and promotes CRC immune surveillance in the tumor stroma. Both of these Tyk2-dependent mechanisms must work together to prevent CRC progression.

Colitis is a major risk factor for the development of colorectal cancer, leading to colitis-associated colorectal cancer (CAC). The most commonly used animal model to study CAC is the azoxymethane-dextran sulphate-sodium (AOM/DSS) model. The ideal experimental conditions of this model depend on several factors, including the used mouse strain. No data on feasibility and conditions for older mice, e.g., for aging studies, have yet been reported. Thus, we conducted a descriptive, observational pilot study where CAC was induced in 14-month-old female Balb/C and C57/Bl6 mice using 12.5 mg/kg AOM i.p. and three different concentrations of DSS (1, 2, and 3%) in drinking water (ad. lib.). The mice were monitored regularly during the three-month experimental phase. After euthanasia, the colons of the mice were evaluated macroscopically and microscopically. Both the mouse strains showed a DSS-concentration-dependent induction of CAC. Carcinomas were only observed at 3% DSS. The DSS dose was found to be significantly correlated with the histology score and % Ki67 positive cells only in C57/Bl6 mice but not in Balb/C mice, which showed a variable response to the CAC induction. No differences in colon length, weight, or mucin content were observed. Optimal conditions for CAC induction in these aged animals are thus considered to be 3% DSS, as carcinomas did not develop when 2% DSS was used. On the other hand, Balb/C mice reacted severely to 3% DSS, indicating that 2.5% DSS may be the “sweet spot” for future experiments comparing CAC in aged Balb/C and C57/Bl6 mice. This model will allow investigation of the effect of aging on CAC development and therapy.

Lung cancer is responsible for the majority of cancer-related deaths worldwide. Within this severe disease lung adenocarcinoma (LUAD) belongs to the most common form of lung cancer. LUAD is highly correlated with KRAS mutations. Although, the first KRAS inhibitors entered into clinics recently, therapy resistance arises. Since KRAS-mutant LUAD is an inflammation driven disease, we focus on the JAK-STAT pathway as an alternative target. Preliminary data suggests that human KRAS-mutant LUAD is correlated to enriched JAK-STAT signaling, as well as STAT1 upregulation. Accordingly, we aimed to explore the tumorigenic functions of STAT1 in this form of lung cancer. Genetically engineered C57BL6/N mice which develop autochthonous KRAS-driven and P53-deficient LUAD proficient (KP) and deficient for STAT1 (KPS) were used. In a second mouse model expression of ovalbumin was induced in tumor cells, mimicking a neoantigen to increase immune infiltration in the presence (KPO) or absence of STAT1 (KPOS). Kaplan-Meyer analysis was performed to assess the overall survival. Tumor burden, as well as immune infiltration was analyzed in lungs 6 and 10 weeks after tumor induction via H&E, IHC and IF staining. Furthermore, the lung fluid was collected by bronchoalveolar lavage and used to profile inflammatory cytokines. Deletion of STAT1 reduced the survival in KRAS-driven LUAD mice only when ovalbumin was expressed. Tumor burden and tumor grades were increased in tumors lacking STAT1 at 6 and 10 weeks after tumor initiation. Surprisingly, although tumors of KPOS mice showed a decreased infiltration by T cells and CD4 T cells, CD8 T cell infiltration did not change, suggesting a tumor suppressive function of STAT1 via T cell exhaustion. Moreover, the macrophage attracting cytokine CCL9 secretion was upregulated and the number of tumor infiltrating, immunosuppressive macrophages was increased in KPOS mice. This data implicates a tumor suppressive function of STAT1 by secretion of cytokines that can recruit suppressive myeloid immune cells to the TME leading to CD4 T cell exclusion and CD8 T cell exhaustion. Further studies are needed on the changes in the immune infiltrating cells to explain the mechanism behind these intriguing findings. Citation Format: Christoph Trenk, Rebecca Sagmeister, Jaqueline Horvath, Monika Homolya, Andreea Corina Luca, Robert Eferl, Herwig Moll, Emilio Casanova. STAT1 suppresses KRAS-driven lung adenocarcinoma depending on the tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3650.

Metastatic colorectal cancer is one of the leading causes of cancer-related deaths worldwide. Traditional chemotherapy extended the lifespan of cancer patients by only a few months, but targeted therapies and immunotherapy prolonged survival and led to long-term remissions in some cases. Type I and II interferons have direct pro-apoptotic and anti-proliferative effects on cancer cells and stimulate anti-cancer immunity. As a result, interferon production by cells in the tumor microenvironment is in the spotlight of immunotherapies as it affects the responses of anti-cancer immune cells. However, promoting effects of interferons on colorectal cancer metastasis have also been reported. Here we summarize our knowledge about pro- and anti-metastatic effects of type I and II interferons in colorectal cancer liver metastasis and discuss possible therapeutic implications.

Tumors have evolved mechanisms to escape anti-tumor immunosurveillance. They limit humoral and cellular immune activities in the stroma and render tumors resistant to immunotherapy. Sensitizing tumor cells to immune attack is an important strategy to revert immunosuppression. However, the underlying mechanisms of immune escape are still poorly understood. Here we discover Indoleamine-2,3-dioxygenase-1 (IDO1)+ Paneth cells in the stem cell niche of intestinal crypts and tumors, which promoted immune escape of colorectal cancer (CRC). Ido1 expression in Paneth cells was strictly Stat1 dependent. Loss of IDO1+ Paneth cells in murine intestinal adenomas with tumor cell-specific Stat1 deletion had profound effects on the intratumoral immune cell composition. Patient samples and TCGA expression data suggested corresponding cells in human colorectal tumors. Thus, our data uncovered an immune escape mechanism of CRC and identify IDO1+ Paneth cells as a target for immunotherapy., Pflügler, Svinka et al. identify a subset of Paneth cells in mouse intestinal crypts and tumors, which express the immune checkpoint molecule Ido1 in a Stat1-dependent manner and promote tumor growth. Gene expression data from human colorectal cancer (CRC) suggest that a similar population is present in human cancer and opens the door for further studies of immune escape mechanisms in CRC.

The canonical WNT signaling pathway is crucial for intestinal stem cell renewal and aberrant WNT signaling is an early event in colorectal cancer (CRC) development. Here, we show for the first time that WNT2 is one of the most significantly induced genes in CRC stroma as compared to normal stroma. The impact of stromal WNT2 on carcinoma formation or progression was not addressed so far. Canonical WNT/β-catenin signaling was assessed using a 7TGP-reporter construct. Furthermore, effects of WNT2 on fibroblast migration and invasion were determined using siRNA-mediated gene silencing. Tumor cell invasion was studied using organotypic raft cultures and in vivo significance was assessed via a xenograft mouse model. We identified cancer-associated fibroblasts (CAFs) as the main source of WNT2. CAF-derived WNT2 activated canonical signaling in adenomatous polyposis coli/β-catenin wild-type colon cancer cells in a paracrine fashion, whereas no hyperactivation was detectable in cell lines harboring mutations in the adenomatous polyposis coli/β-catenin pathway. Furthermore, WNT2 activated autocrine canonical WNT signaling in primary fibroblasts, which was associated with a pro-migratory and pro-invasive phenotype. We identified FZD8 as the putative WNT2 receptor in CAFs. Three-dimensional organotypic co-culture assays revealed that WNT2-mediated fibroblast motility and extracellular matrix remodeling enhanced cancer cell invasion of cell lines even harboring mutations in the adenomatous polyposis coli/β-catenin pathway. Thus, suggesting a tumor-promoting influence on a broad range of CRC. In line, WNT2 also promotes tumor growth, invasion and metastasis in vivo. Moreover, high WNT2 expression is associated with poor prognosis in human CRC. The identification of the pro-malignant function of stromal derived WNT2 in CRC classifies WNT2 and its receptor as promising stromal targets to confine cancer progression in combination with conventional or targeted therapies.

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.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

TYK2 is a member of the JAK family of tyrosine kinases that is involved in chromosomal translocation-induced fusion proteins found in anaplastic large cell lymphomas (ALCL) that lack rearrangements activating the anaplastic lymphoma kinase (ALK). Here we demonstrate that TYK2 is highly expressed in all cases of human ALCL, and that in a mouse model of NPM-ALK-induced lymphoma, genetic disruption of Tyk2 delays the onset of tumors and prolongs survival of the mice. Lymphomas in this model lacking Tyk2 have reduced STAT1 and STAT3 phosphorylation and reduced expression of Mcl1, a pro-survival member of the BCL2 family. These findings in mice are mirrored in human ALCL cell lines, in which TYK2 is activated by autocrine production of IL-10 and IL-22 and by interaction with specific receptors expressed by the cells. Activated TYK2 leads to STAT1 and STAT3 phosphorylation, activated expression of MCL1 and aberrant ALCL cell survival. Moreover, TYK2 inhibitors are able to induce apoptosis in ALCL cells, regardless of the presence or absence of an ALK-fusion. Thus, TYK2 is a dependency that is required for ALCL cell survival through activation of MCL1 expression. TYK2 represents an attractive drug target due to its essential enzymatic domain, and TYK2-specific inhibitors show promise as novel targeted inhibitors for ALCL.

Oncogenic K‐RAS has been difficult to target and currently there is no K‐RAS‐based targeted therapy available for patients suffering from K‐RAS‐driven lung adenocarcinoma (AC). Alternatively, targeting K‐RAS‐downstream effectors, K‐RAS‐cooperating signaling pathways or cancer hallmarks, such as tumor‐promoting inflammation, has been shown to be a promising therapeutic strategy. Since the JAK–STAT pathway is considered to be a central player in inflammation‐mediated tumorigenesis, we investigated here the implication of JAK–STAT signaling and the therapeutic potential of JAK1/2 inhibition in K‐RAS‐driven lung AC. Our data showed that JAK1 and JAK2 are activated in human lung AC and that increased activation of JAK–STAT signaling correlated with disease progression and K‐RAS activity in human lung AC. Accordingly, administration of the JAK1/2 selective tyrosine kinase inhibitor ruxolitinib reduced proliferation of tumor cells and effectively reduced tumor progression in immunodeficient and immunocompetent mouse models of K‐RAS‐driven lung AC. Notably, JAK1/2 inhibition led to the establishment of an antitumorigenic tumor microenvironment, characterized by decreased levels of tumor‐promoting chemokines and cytokines and reduced numbers of infiltrating myeloid derived suppressor cells, thereby impairing tumor growth. Taken together, we identified JAK1/2 inhibition as promising therapy for K‐RAS‐driven lung AC., What's new? A drug that inhibits the JAK–STAT pathway may score a hit against K‐RAS driven lung cancer. Here, the authors Investigated the JAK STAT pathway as a possible target in lung adenocarcinoma because of its role in inflammation‐mediated tumorigenesis. First, they showed that JAK1 and JAK2 are both activated in lung adenocarcinoma patients with oncogenic mutations in K‐RAS. Next, they treated the tumors with ruxolitinib, which inhibits JAK1/2. The drug successfully slowed tumor proliferation and progression in immunocompetent mouse models. Furthermore, treatment with ruxolitinib reduced the tumor‐promoting factors present in the microenvironment.

The interleukin (IL)-1 family of cytokines is strongly associated with systemic sclerosis (SSc) and pulmonary involvement, but the molecular mechanisms are poorly understood. The aim of this study was to assess the role of IL-1α and IL-1β in pulmonary vascular and interstitial remodelling in a mouse model of SSc. IL-1α and IL-1β were localised in lungs of SSc patients and in the fos-related antigen-2 (Fra-2) transgenic (TG) mouse model of SSc. Lung function, haemodynamic parameters and pulmonary inflammation were measured in Fra-2 TG mice with or without 8 weeks of treatment with the IL-1 receptor antagonist anakinra (25 mg·kg−1·day−1). Direct effects of IL-1 on pulmonary arterial smooth muscle cells (PASMCs) and parenchymal fibroblasts were investigated in vitro. Fra-2 TG mice exhibited increased collagen deposition in the lung, restrictive lung function and enhanced muscularisation of the vasculature with concomitant pulmonary hypertension reminiscent of the changes in SSc patients. Immunoreactivity of IL-1α and IL-1β was increased in Fra-2 TG mice and in patients with SSc. IL-1 stimulation reduced collagen expression in PASMCs and parenchymal fibroblasts via distinct signalling pathways. Blocking IL-1 signalling in Fra-2 TG worsened pulmonary fibrosis and restriction, enhanced T-helper cell type 2 (Th2) inflammation, and increased the number of pro-fibrotic, alternatively activated macrophages. Our data suggest that blocking IL-1 signalling as currently investigated in several clinical studies might aggravate pulmonary fibrosis in specific patient subsets due to Th2 skewing of immune responses and formation of alternatively activated pro-fibrogenic macrophages., IL-1 dampens collagen production of lung structural cells and balances pro-fibrotic actions of the immune system. Blockade of IL-1 signalling in Fra-2 TG mice worsens lung function by increased Th2 inflammation and collagen production in the lung. http://bit.ly/2IVUGLX

Tumor-infiltrating immune cells are highly relevant for prognosis and identification of immunotherapy targets in hepatocellular carcinoma (HCC). The recently developed CIBERSORT method allows immune cell profiling by deconvolution of gene expression microarray data. By applying CIBERSORT, we assessed the relative proportions of immune cells in 41 healthy human livers, 305 HCC samples and 82 HCC adjacent tissues. The obtained immune cell profiles provided enumeration and activation status of 22 immune cell subtypes. Mast cells were evaluated by immunohistochemistry in ten HCC patients. Activated mast cells, monocytes and plasma cells were decreased in HCC, while resting mast cells, total and naïve B cells, CD4+ memory resting and CD8+ T cells were increased when compared to healthy livers. Previously described S1, S2 and S3 molecular HCC subclasses demonstrated increased M1-polarized macrophages in the S3 subclass with good prognosis. Strong total immune cell infiltration into HCC correlated with total B cells, memory B cells, T follicular helper cells and M1 macrophages, whereas weak infiltration was linked to resting NK cells, neutrophils and resting mast cells. Immunohistochemical analysis of patient samples confirmed the reduced frequency of mast cells in human HCC tumor tissue as compared to tumor adjacent tissue. Our data demonstrate that deconvolution of gene expression data by CIBERSORT provides valuable information about immune cell composition of HCC patients.

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. (Hepatology 2015;61:613-626)

The interferon‐inducible transcription factor STAT1 is a tumor suppressor in various malignancies. We investigated sex‐specific STAT1 functions in colitis and colitis‐associated colorectal cancer (CRC) using mice with specific STAT1 deletion in intestinal epithelial cells (STAT1∆IEC). Male but not female STAT1∆IEC mice were more resistant to DSS‐induced colitis than sex‐matched STAT1flox/flox controls and displayed reduced intraepithelial infiltration of CD8+ TCRαβ+ granzyme B+ T cells. Moreover, DSS treatment failed to induce expression of T‐cell‐attracting chemokines in intestinal epithelial cells of male but not of female STAT1∆IEC mice. Application of the AOM‐DSS protocol for induction of colitis‐associated CRC resulted in increased intestinal tumor load in male but not in female STAT1∆IEC mice. A sex‐specific stratification of human CRC patients corroborated the data obtained in mice and revealed that reduced tumor cell‐intrinsic nuclear STAT1 protein expression is a poor prognostic factor in men but not in women. These data demonstrate that epithelial STAT1 is a male‐specific tumor suppressor in CRC of mice and humans.

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.

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.

The German cytologist Theodor Boveri proposed about 100 years ago that cancer is caused by an abnormal distribution of microscopic bodies called chromosomes. At that time, researchers had no idea of the cellular component for inheritance and challenged his hypothesis. Meanwhile, genomic instability is viewed as a hallmark of cancer1 and an alternative model for cancer formation was put forward that proposed acquisition of a ‘mutator’ phenotype as the initiating event. Although supported by the vast plasticity of sequence changes and chromosomal aberrations in individual cells of single tumours, these observations did not solve the issue whether the ‘mutator’ phenotype is a precondition or a consequence of tumourigenesis. The ‘mutator’ phenotype is causative for genomic instability, an umbrella term for small DNA structure variations, microsatellite instability and chromosomal instability (CIN).2 The latter affects chromosome number and structure and is a characteristic feature of many cancer types including hepatocellular carcinoma (HCC).3 It is also associated with the formation of extranuclear bodies that contain damaged chromosome fragments or whole chromosomes. Such micronuclei were identified in regenerative and dysplastic nodules of the liver indicating that CIN can be acquired already in early stage hepatocarcinogenesis.3 Apparently, CIN is linked to certain cancer genotypes because HCC has been originally divided into two major subclasses: one displaying a stable cancer genome and activating β-catenin mutations, and the other with extensive CIN, p53 and Axin …

Vascular remodelling is a hallmark of pulmonary hypertension (PH) and is characterized by enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs). Accumulating evidence indicates a crucial role of transcription factors in the vascular remodelling processes. Here, we characterize the involvement of meprin β, a novel activator protein-1 (AP-1) effector molecule, in PH. Fra-2 transgenic (TG) mice exhibited increased right ventricular systolic pressure (RVSP), accompanied by vascular remodelling and activation of the pro-proliferative and pro-fibrotic AKT pathway. Microarray studies revealed the collagen-processing metalloprotease meprin β as the most up-regulated gene in Fra-2 TG mice. Its expression, increased at all investigated time points, preceded the decreased expression of MMPs and increased TGFβ, followed by collagen deposition. Correspondingly, remodelled pulmonary arteries from explanted idiopathic pulmonary arterial hypertension (IPAH) patients' lungs exhibited pronounced expression of meprin β. Fra-2 and meprin β expression in human PASMCs was regulated by PDGF-BB and TGFβ in a complementary fashion. Importantly, PDGF-BB-dependent proliferation was attenuated by silencing AP-1 expression or by meprin β inhibition. This study delineates a novel molecular mechanism underlying PASMCs proliferation and extracellular matrix (ECM) deposition by identifying meprin β as an important mediator in regulating vascular remodelling processes. Thus, meprin β may represent a new molecule that can be targeted in pulmonary hypertension. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Activation of the activator protein 1 (AP-1) transcription factor as well as increased serum levels of vascular endothelial growth factor (VEGF) and interleukin (IL)-8 predict poor prognosis of patients with hepatocellular carcinomas (HCCs). Moreover, HCC patients display reduced selenium levels, which may cause lipid peroxidation and oxidative stress because selenium is an essential component of antioxidative glutathione peroxidases (GPx). We hypothesized that selenium-lipid peroxide antagonism controls the above prognostic markers and tumor growth. (1) In human HCC cell lines (HCC-1.2, HCC-3, and SNU398) linoleic acid peroxide (LOOH) and other prooxidants enhanced the expression of VEGF and IL-8. LOOH up-regulated AP-1 activation. Selenium inhibited these effects. This inhibition was mediated by glutathione peroxidase 4 (GPx4), which preferentially degrades lipid peroxides. Selenium enhanced GPx4 expression and total GPx activity, while knock-down of GPx4 by small interfering RNA (siRNA) increased VEGF, and IL-8 expression. (2) These results were confirmed in a rat hepatocarcinogenesis model. Selenium treatment during tumor promotion increased hepatic GPx4 expression and reduced the expression of VEGF and of the AP-1 component c-fos as well as nodule growth. (3) In HCC patients, increased levels of LOOH-related antibodies (LOOH-Ab) were found, suggesting enhanced LOOH formation. LOOH-Ab correlated with serum VEGF and IL-8 and with AP-1 activation in HCC tissue. In contrast, selenium inversely correlated with VEGF, IL-8, and HCC size (the latter only for tumors smaller than 3 cm). Conclusion: Reduced selenium levels result in accumulation of lipid peroxides. This leads to enhanced AP-1 activation and consequently to elevated expression of VEGF and IL-8, which accelerate the growth of HCC. Selenium supplementation could be considered for investigation as a strategy for chemoprevention or additional therapy of early HCC in patients with low selenium levels. (HEPATOLOGY 2012)

Tumor invasion and metastasis is a multi-step process that requires adaptation of cancer cells to conditions that they encounter during their journey to distant body sites. Understanding the molecular processes that underlie this adaptation is of exceeding importance because most cancer patients die because of metastases rather than primary tumors. In this review we assess genetically engineered mouse models (GEMMs) that have been established to investigate mechanisms of cancer invasion and metastasis.

Background & Aims Signal transducer and activator of transcription 3 (Stat3) is the main mediator of interleukin-6–type cytokine signaling required for hepatocyte proliferation and hepatoprotection, but its role in sclerosing cholangitis and other cholestatic liver diseases remains unresolved. Methods We investigated the role of Stat3 in inflammation-induced cholestatic liver injury and used mice lacking the multidrug resistance gene 2 (mdr2−/−) as a model for SC. Results We show that conditional inactivation of Stat3 in hepatocytes and cholangiocytes (stat3Δhc) of mdr2−/− mice strongly aggravated bile acid–induced liver injury and fibrosis. A similar phenotype was observed in mdr2−/− mice lacking interleukin-6 production. Biochemical and molecular characterization suggested that Stat3 exerts hepatoprotective functions in both hepatocytes and cholangiocytes. Loss of Stat3 led to increased expression of tumor necrosis factor α, which might reduce the barrier function of bile ducts. Moreover, Stat3-deficient hepatocytes displayed up-regulation of bile acid biosynthesis genes and down-regulation of hepatoprotective epidermal growth factor receptor and insulin-like growth factor 1 signaling pathways. Consistently, stat3Δhc mice were more sensitive to cholic acid–induced liver damage than control mice. Conclusions Our data suggest that Stat3 prevents cholestasis and liver damage in sclerosing cholangitis via regulation of pivotal functions in hepatocytes and cholangiocytes.

Growth hormone (GH) resistance and low serum levels of insulinlike growth factor 1 (IGF-1) are common features in human liver fibrosis and cirrhosis. Signal transducer and activator of transcription 5 (STAT5) controls several vital functions in the liver, including GH-mediated transcription of IGF-1. To investigate the role of STAT5 in liver fibrogenesis, we specifically deleted the Stat5a/b locus both in hepatocytes and cholangiocytes in the multidrug resistance gene 2 knockout (Mdr2−/−) mouse model of cholestasis. Double knockout mice develop an early and severe liver fibrosis phenotype, accompanied by perturbed expression of key regulators of bile acid homeostasis. Deletion of Stat5 resulted in GH resistance, and IGF-1 levels in serum were undetectable. We could observe reduced expression of important hepatoprotective genes, such as epidermal growth factor receptor (Egfr), hepatocyte nuclear factor 6 (Hnf6), prolactin receptor (Prlr), and leukemia inhibitory factor receptor (Lifr) as well as increased numbers of apoptotic hepatocytes. Conclusion Our data suggest that loss of STAT5 sensitizes hepatocytes to bile acid–induced damage and apoptosis caused by disruption of GH-induced transcription of Igf-1 and down-regulation of hepatoprotective genes. These findings could contribute to the understanding of liver fibrosis and future treatment strategies for liver fibrosis.

The use of bacterial artificial chromosomes (BACs) modified via homologous recombination in Escherichia coli has become a powerful tool in the transgenic field. Homologous recombination allows the manipulation of BACs in very different ways. However, this process can be cumbersome and problematic when using large targeting constructs containing several repeated elements. In order to address this problem, we have established a ΦC31 integrase-mediated cassette exchange into a BAC. As an example of this technique, we have exchanged a cassette previously recombined into a BAC containing the Rosa 26 locus, by a 16.5-kb incoming construct containing several repeated elements. The combination of homologous recombination in E. coli and cassette exchange should expand the tools for manipulating BACs, thus facilitating the generation of constructs with higher complexity.

Bone resorbing osteoclasts are specialized macrophages that cannot differentiate in the absence of c-Fos, a member of the dimeric transcription factor AP-1 (activator protein-1). However, osteoclast differentiation in the absence of c-Fos can be rescued in vitro and in vivo by Fra1, a Fos-like protein and transcriptional target of c-Fos. To enable AP-1 proteins binding to DNA, c-Fos or Fra1 must heterodimerize with a partner such as c-Jun, JunB and JunD. In this study, we investigated the dimerization partners of c-Fos and Fra1 required for osteoclast differentiation using synthetic "single-chain" AP-1 dimers in which c-Fos or Fra1 is tethered via a linker to Jun proteins. When c-Fos was analyzed in combination with any Jun protein, including a c-Jun mutant lacking major phosphorylation sites for c-Jun amino-terminal kinase (JNK), osteoclasts were efficiently formed from c-Fos-deficient hematopoietic precursors. However, Fra1 in combination with any Jun protein could not rescue osteoclastogenesis. The ability to rescue was compared to transcriptional activity measured in transient transfection assays using promoters driven by consensus AP-1 sites or a composite AP-1/NFAT binding site. These data show that a single Jun/c-Fos dimer is sufficient for osteoclast differentiation, likely due to its transactivation ability for a broader range of promoters, in particular consensus AP-1 sites. We propose that Fra1 together with a dimerization partner different from Jun proteins can rescue osteoclast differentiation in c-Fos-deficient precursors.

Loss of function mouse models comprise knock-out mice, where a gene is deleted in the germline, and conditional knock-out mice with somatic deletion of a floxed allele in defined tissues. Both types of mice are used for comprehensive studies of gene functions in vivo. Here, we describe a simple method for simultaneous generation of mice with conditional or knock-out alleles for the transcription factor fra-2 (Fos-related antigen 2) using a single embryonic stem (ES) cell clone. ES cells with a floxed fra-2 allele were transiently transfected with a Cre-recombinase expression plasmid and plated at low density. Most of the resulting ES cell colonies consisted of a mixture of cells that have either retained or lost the conditional allele. We demonstrate that these mixed ES cell clones can be directly used for generation of chimeras that give rise to offspring with conditional or knock-out alleles simultaneously. This strategy shortens the time and reduces the number of germline transmission events to generate genetically modified mice.

The AP-1 transcription factor c-Jun is a key regulator of hepatocyte proliferation. Mice lacking c-Jun in the liver (c-junΔli*) display impaired liver regeneration after partial hepatectomy (PH). This phenotype correlates with increased protein levels of the cdk-inhibitor p21 in the liver. We performed PH experiments in several double-knockout mouse models to genetically identify the signaling events regulated by c-Jun. Inactivation of p53 in c-junΔli* mice abrogated both hepatocyte cell cycle block and increased p21 protein expression. Consistently, liver regeneration was rescued in c-junΔli*p21−/− double-mutant mice. This indicated that c-Jun controls hepatocyte proliferation by a p53/p21-dependent mechanism. Analyses of p21 mRNA and protein expression in livers of c-junΔli* mice after PH revealed that the accumulation of p21 protein is due to a post-transcriptional/post-translational mechanism. We have investigated several candidate pathways implicated in the regulation of p21 expression, and observed increased activity of the stress kinase p38 in regenerating livers of c-junΔli* mice. Importantly, conditional deletion of p38α in livers of c-junΔli* mice fully restored hepatocyte proliferation and attenuated increased p21 protein levels after PH. These data demonstrate that c-Jun/AP-1 regulates liver regeneration through a novel molecular pathway that involves p53, p21, and the stress kinase p38α.

Hepatocytes are the foreign legionnaires in our body. They are exposed to various xenobiotics, which invigorates them and when transformed into malignant cells, renders them drug resistant. So far, treatment with the multikinase inhibitor sorafenib, which blocks Raf, vascular endothelial growth factor receptor and platelet-derived growth factor receptor signalling, is the only approved targeted therapy for advanced hepatocellular carcinoma (HCC). However, sorafenib extends the average life expectancy of patients only 2.8 months over the placebo,1 which offers room for improvement. Unfortunately, several other drugs and combinatorial therapies, such as sorafenib plus erlotinib failed in clinical trials.1 ,2 In this issue of Gut , Bollard et al 3 tested the combination of sorafenib with palbociclib in preclinical HCC mouse models. Palbociclib (or PD0332991) is a reversible and orally bioavailable low-molecular-weight cyclin-dependent kinase (CDK)4/6 inhibitor without significant off-target effects.4 The history of palbociclib is tightly linked to the decipherment of G1 cell cycle checkpoint regulation that started in 1991 with the discovery of D-type cyclins. CDK4 and CDK6 were discovered soon thereafter. They bind physically to and are enzymatically activated by D-type cyclins, which leads to phosphorylation and inactivation of retinoblastoma (RB1), the key gatekeeper of G1/S transition. Given the importance RB1 in cancer development, pharmacists started to develop CDK4/6 inhibitors such as palbociclib (Pfizer), ribociclib (Novartis) and abemaciclib (Elli Lilly). However, early clinical trials with palbociclib monotherapy dampened the initial enthusiasm for this drug, although tumour regression was observed in some patients with liposarcoma. A breakthrough was achieved when palbociclib was combined with letrozole, an aromatase inhibitor, for the treatment of oestrogen receptor-positive breast …

Because JunB is an essential gene for placentation, it was conditionally deleted in the embryo proper. JunBDelta/Delta mice are born viable, but develop severe low turnover osteopenia caused by apparent cell-autonomous osteoblast and osteoclast defects before a chronic myeloid leukemia-like disease. Although JunB was reported to be a negative regulator of cell proliferation, junBDelta/Delta osteoclast precursors and osteoblasts show reduced proliferation along with a differentiation defect in vivo and in vitro. Mutant osteoblasts express elevated p16(INK4a) levels, but exhibit decreased cyclin D1 and cyclin A expression. Runx2 is transiently increased during osteoblast differentiation in vitro, whereas mature osteoblast markers such as osteocalcin and bone sialoprotein are strongly reduced. To support a cell-autonomous function of JunB in osteoclasts, junB was inactivated specifically in the macrophage-osteoclast lineage. Mutant mice develop an osteopetrosis-like phenotype with increased bone mass and reduced numbers of osteoclasts. Thus, these data reveal a novel function of JunB as a positive regulator controlling primarily osteoblast as well as osteoclast activity.

The Fos protein, a major component of the AP-1 transcription factor, is essential for osteoclast differentiation, acts as an oncogene, potentiates transforming signals, and controls invasive growth and angiogenesis during tumor progression. To investigate a potential genetic interaction between the Trp53 and Fos pathways, Trp53/Fos double knockout mice were generated. These mice develop highly proliferative and invasive rhabdomyosarcomas of the facial and orbital regions, with more than 90% penetrance at 6 months of age. Rhabdomyosarcoma cell lines established from the primary tumors express characteristic muscle-specific markers, and reexpression of Fos is associated with enhanced apoptosis in vitro. Moreover, Fos is able to repress Pax7 expression in rhabdomyosarcoma cell lines and primary myoblasts, suggesting a molecular link to genetic alterations involved in human rhabdomyosarcomas.

To investigate the function of c-Jun during skin development and skin tumor formation, we conditionally inactivated c-jun in the epidermis. Mice lacking c-jun in keratinocytes (c-jun(Deltaep)) develop normal skin but express reduced levels of EGFR in the eyelids, leading to open eyes at birth, as observed in EGFR null mice. Primary keratinocytes from c-jun(Deltaep) mice proliferate poorly, show increased differentiation, and form prominent cortical actin bundles, most likely because of decreased expression of EGFR and its ligand HB-EGF. In the absence of c-Jun, tumor-prone K5-SOS-F transgenic mice develop smaller papillomas, with reduced expression of EGFR in basal keratinocytes. Thus, using three experimental systems, we show that EGFR and HB-EGF are regulated by c-Jun, which controls eyelid development, keratinocyte proliferation, and skin tumor formation.

The transcription factor c-Jun mediates several cellular processes, including proliferation and survival, and is upregulated in many carcinomas. Liver-specific inactivation of c-Jun at different stages of tumor development was used to study its role in chemically induced hepatocellular carcinomas (HCCs) in mice. The requirement for c-jun was restricted to early stages of tumor development, and the number and size of hepatic tumors was dramatically reduced when c-jun was inactivated after the tumor had initiated. The impaired tumor development correlated with increased levels of p53 and its target gene noxa, resulting in the induction of apoptosis without affecting cell proliferation. Primary hepatocytes lacking c-Jun showed increased sensitivity to TNF-α-induced apoptosis, which was abrogated in the absence of p53. These data indicate that c-Jun prevents apoptosis by antagonizing p53 activity, illustrating a mechanism that might contribute to the early stages of human HCC development.

Colorectal cancer (CRC) originates from the epithelial cells lining the colon or rectum of the gastrointestinal tract and represents the third most common form of cancer worldwide. CRC is frequently associated with Colitis Ulcerosa or Crohn's Disease demonstrating the tumor-promoting role of inflammation. Colorectal tumor cells establish heterotypic interactions with inflammatory cells and cancer-associated fibroblasts in the tumor stroma that support tumor angiogenesis and are essential for tumor progression. Therefore, establishment of suitable mouse models mimicking the inflammatory etiology of CRC is important. Here we describe methods to induce CRC in mice, to quantify tumor parameters (multiplicity, tumor load, mean tumor size), and to analyze the cellular composition of the CRC tumor stroma.

Colorectal cancer (CRC) originates from the epithelial cells lining the colon or rectum of the gastrointestinal tract and represents the third most common form of cancer worldwide. CRC is frequently associated with Colitis Ulcerosa or Crohn's Disease demonstrating the tumor-promoting role of inflammation. Colorectal tumor cells establish heterotypic interactions with inflammatory cells and cancer-associated fibroblasts in the tumor stroma that support tumor angiogenesis and are essential for tumor progression. Therefore, establishment of suitable mouse models mimicking the inflammatory etiology of CRC is important. Here we describe methods to induce CRC in mice, to quantify tumor parameters (multiplicity, tumor load, mean tumor size), and to analyze the cellular composition of the CRC tumor stroma.

Straight-chain acyl-CoA oxidase is the first and rate limiting enzyme in the peroxisomal β-oxidation pathway catalysing the desaturation of acyl-CoAs to 2-trans-enoyl-CoAs, thereby producing H2O2. To study peroxisomal β-oxidation we cloned and characterized the cDNA of mouse peroxisomal acyl-CoA oxidase. It consists of 3778 bp, including a 1983-bp ORF encoding a polypeptide of 661 amino-acid residues. Like the rat and human homologue the C-terminus contains an SKL motif, an import signal present in several peroxisomal matrix proteins. Sequence analysis revealed high amino-acid homology with rat (96%) and human (87%) acyl-CoA oxidase in addition to minor homology (≈ 40%) with other related proteins, such as rabbit trihydroxy-cholestanoyl-CoA oxidase, human branched chain acyl-CoA oxidase and rat trihydroxycoprostanoyl-CoA oxidase. Acyl-CoA oxidase mRNA and protein expression were most abundant in liver followed by kidney, brain and adipose tissue. During mouse brain development acyl-CoA oxidase mRNA expression was highest during the suckling period indicating that peroxisomal β-oxidation is most critical during this developmental stage. Comparing tissue mRNA levels of peroxisome proliferator-activated receptor alpha and acyl-CoA oxidase, we noticed a constant relationship in all tissues investigated, except heart and adipose tissue in which much more, and respectively, much less, peroxisome proliferator-activated receptor alpha mRNA in proportion to acyl-CoA oxidase mRNA was found. Our data show that acyl-CoA oxidase is an evolutionary highly conserved enzyme with a distinct pattern of expression and indicate an important role in lipid metabolism.

The proto-oncogene c-jun encodes a component of the transcription factor AP-1 (activating protein 1)1, which has been implicated in the regulation of diverse cellular functions, such as proliferation, differentiation, transformation, and apoptosis. AP-1 is a dimer consisting of different subunits, e.g., proteins of the Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB, Fra1, and Fra2) family as well as CREB/ATF, and Maf proteins. The different AP-1 components are expressed in a development- and tissue-specific manner, implying that AP-1 composed of different subunits may exert different functions in different cell types. Although AP-1 was found to regulate a few genes, such as human metallothionein IIA (Lee et al., 1987), collagenase (Angel et al., 1987), stromelysin (Kerr et al., 1988), and keratin 18 (Oshima et al., 1990), the biological function of the different AP-1 complexes during development is still elusive. The characterization of the role of AP-1 is further impeded by the fact that there are, in addition to the variability in subunit composition, numerous possible interactions between AP-1 and other transcription factors, such as glucocorticoid hormone receptors (Jonat et al., 1990), estrogen receptors (Gaub et al., 1990), retinoic acid and vitamin D3 receptors (Schule et al., 1990), and MyoD (Bengal et al., 1992) yielding a network of transcriptional regulation. First clues on tissue-specific functions of AP-1 components came from gene knockout experiments. In c-fos knockout mice the development of bone is impaired because of a block in osteoclast differentiation (Grigoriadis et al., 1994). Moreover, lymphoid cells, germ cells, and neuronal tissues are affected in the absence of c-Fos (Johnson et al., 1992; Wang et al., 1992). In contrast to the inactivation of c-fos, targeted disruption of c-jun and junB is lethal (Hilberg et al., 1993; Johnson et al., 1993; Schorpp-Kistner et al., 1999). Lethality of c-jun−/− fetuses has been suggested to be due to defective liver development. The livers of some E12.5 animals appeared hypoplastic with rounded, dissociated hepatoblasts showing features of apoptosis and necrosis. Moreover, increased numbers of erythropoietic cells were noted in these livers (Hilberg et al., 1993). A defect in hepatogenesis in c-Jun knockout mice was further indicated by the observation that c-jun−/− embryonic stem (ES) cells failed to contribute to the liver but not to other tissues of adult chimeric mice (Hilberg et al., 1993). These observations, together with the fact that no morphological alterations were found in organs other than the liver, led to the conclusion that the absence of c-Jun might preferentially affect the development of the liver (Hilberg et al., 1993). In the mouse, liver development starts at around E9.5 when epithelial cells of the foregut endoderm proliferate and invade the mesenchyme of the septum transversum thus forming the embryonic liver. At around E11 hematopoietic stem and progenitor cells derived from the yolk sac and aorta-gonad-mesonephros region colonize the liver, and the liver becomes the major hematopoietic organ during further fetal development (Dzierzak and Medvinsky, 1995). To allow establishment and maintenance of hematopoiesis, liver cells have to provide the proper microenvironment for hematopoietic cells comparable to stromal cells in the bone marrow during postnatal life. The next major step in mouse liver development occurs at approximately E14.5 when hepatoblasts start to differentiate into the hepatocytic and bile duct epithelial lineage, which is indicated by the formation of the ductal plate, which later differentiates into the intrahepatic bile ducts (Desmet, 1998). It is as yet unclear at which developmental stage c-Jun becomes essential for the liver, and whether the defect is restricted to the hepatocytic lineage or other cell types of the fetal liver, such as bile duct epithelia, endothelial cells, stellate cells (vitamin A–storing cells), Kupffer cells, and hematopoietic cells. Besides the poorly characterized function in liver development, c-Jun plays a more general role in the regulation of cell proliferation and apoptosis. It has been shown in fibroblasts isolated from E11.5 c-jun−/− and c-jun+/− embryos that the absence or diminished expression of c-Jun resulted in greatly reduced growth rates, and that this proliferation defect could not be compensated by addition of purified mitogens (Johnson et al., 1993; Schreiber et al., 1999). Evidence for a role of c-Jun and c-Jun phosphorylation in apoptosis was obtained in neuronal cells where transient overexpression of c-Jun induced apoptosis, and expression of a dominant negative c-jun mutant inhibited apoptosis in vitro (Estus et al., 1994; Ham et al., 1995; Behrens et al., 1999). In vivo, however, c-Jun was regarded not to be essential for apoptosis since in the developing mouse (E11.5 c-jun−/− fetuses) the physiologically occurring apoptosis appeared unaffected (Roffler-Tarlov et al., 1996). The different phenotypes observed in the various AP-1 knockout mice point to cell type– and developmental-specific roles of AP-1 complexes. The biological basis for the specific roles is not yet understood. To gain more insight into how the absence of a widely expressed transcription factor like c-Jun affects the liver, and to see whether other tissues are affected, we investigated in detail the morphological and functional alterations in c-jun knockout mice as well as the distribution of c-jun−/− cells in chimeric mice at various stages during fetal development and postnatal life. A deregulation of apoptosis was found in a variety of cell types lacking c-jun, such as hepatoblasts, erythroid cells, and fibroblasts. In contrast to previous reports that suggested that c-Jun is essential for cells to undergo apoptosis, we observed markedly increased apoptotic rates in the absence of c-Jun. It is possible that an increased susceptibility of cells to apoptosis was responsible for the morphologic alterations seen in the livers of c-jun−/− mice. Increased apoptotic rates in combination with reduced proliferation rates would result in a disturbance of hepatocyte turnover which could explain the absence of c-jun−/− hepatocytes in livers of adult chimeric mice. Furthermore, a novel function of c-Jun in heart development was identified, since all c-jun−/− fetuses had a malformation of the outflow tract of the heart which could be a contributing factor to the fetal lethality.

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 KrasG12D-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.

Iron deficiency is a common cause of reactive thrombocytosis, however, the exact pathways have not been revealed. Here we aimed to study the mechanisms behind iron deficiency‐induced thrombocytosis. Within few weeks, iron‐depleted diet caused iron deficiency in young Sprague–Dawley rats, as reflected by a drop in hemoglobin, mean corpuscular volume, hepatic iron content and hepcidin mRNA in the liver. Thrombocytosis established in parallel. Moreover, platelets produced in iron deficient animals displayed a higher mean platelet volume and increased aggregation. Bone marrow studies revealed subtle alterations that are suggestive of expansion of megakaryocyte progenitors, an increase in megakaryocyte ploidy and accelerated megakaryocyte differentiation. Iron deficiency did not alter the production of hematopoietic growth factors such as thrombopoietin, interleukin 6 or interleukin 11. Megakaryocytic cell lines grown in iron‐depleted conditions exhibited reduced proliferation but increased ploidy and cell size. Our data suggest that iron deficiency increases megakaryopoietic differentiation and alters platelet phenotype without changes in megakaryocyte growth factors, specifically TPO. Iron deficiency‐induced thrombocytosis may have evolved to maintain or increase the coagulation capacity in conditions with chronic bleeding. Am. J. Hematol. 89:524–529, 2014. © 2014 Wiley Periodicals, Inc.

p21Waf1/Cip1 was originally identified as a crucial p53-regulated factor that controls cyclin-dependent kinase 1/2 (CDK1/CDK2) activities and sequesters proliferating cell nuclear antigen. The physical interaction with CDKs prevents binding and phosphorylation of retinoblastoma proteins which is required for the release of E2F transcription factors important for S-phase progression. Consequently, p21Waf1/Cip1 was classified as a tumour suppressor that interferes with cell cycle progression and DNA synthesis.1 ,2 Meanwhile, it has been realised that p21Waf1/Cip1 has a plethora of alternative functions in apoptosis, differentiation, migration, DNA repair, gene regulation, stem cell renewal and oncogenesis.1 ,2 Most of these functions are regulated in a dual and p53-independent manner. Cell proliferation is not exclusively inhibited but can also be promoted by p21Waf1/Cip1 when it is present at certain threshold concentrations. During apoptosis, the cytostatic effect of p21Waf1/Cip1 can protect cells from genotoxic insults (which require cell cycle progression for induction of cell death) or through mechanisms that are based on cytoplasmic interference with caspase activation, suppression of E2F1- or Myc-induced pro-apoptotic gene expression and inhibition of pro-apoptotic stress kinases. Alternatively, p21Waf1/Cip1 can induce apoptosis through p53-dependent or -independent regulation of Bax and death receptors.1 ,2 p21Waf1/Cip1 is not readily mutated in human cancers and expression is downregulated …

In the current study we are investigating the effects of PTEN-deficient myeloid cells on tumor immune surveillance. We could previously show that hyper-activation of the PI3K signaling cascade by genetic knock-out of the counteracting phosphatase PTEN induced an anti-inflammatory phenotype in myeloid cells. This resulted in protection of conditional knock-out mice in models of acute infection and inflammation. A reduction in pro-inflammatory responses could however increase tumor burden. To address this question we induced colitis associated colon cancer in conditional PTEN-KO mice and found an increase in tumor burden and a reduction in survival in male KO mice. This was accompanied by increased numbers of splenic antigen-presenting cells (APC) expressing the immune checkpoint regulators PD-L1 and PD-L2. Furthermore, transcriptome analysis in these cells revealed a shift towards gene expression profiles found in professional APCs capable of cross-presentation. As expected, ex-vivo stimulated T-cells from KO-mice showed a reduction in proliferative capacity. These findings were further substantiated by findings in a second tumor model using implanted B16 melanoma cells. In this model myeloid PTEN-deficient mice showed a decrease in T-cell activation and a reduction in melanoma cell killing capacity. Taken together, our findings show that genetic deletion of PTEN in cells of myeloid origin increases splenic APCs expressing immune checkpoint regulators resulting in a decrease in tumor immune surveillance. Our study shows that PI3K-inhibitors which are currently tested as anti-cancer drugs might have additional beneficial effects on immune cells by shifting their inflammatory phenotype. Citation Format: Mario Kuttke, Emine Sahin, Julia Pisoni, Sophie Percig, Andrea Vogel, Daniel Kraemmer, Leslie Hanzl, Julia Stefanie Brunner, Hannah Paar, Klara Soukup, Angela Halfmann, Alexander Dohnal, Carl-Walter Steiner, Stephan Blüml, Jose Basilio, Bernhard Hochreiter, Manuel Salzmann, Bastian Hoesel, Günther Lametschwandtner, Robert Eferl, Johannes Schmid, Gernot Schabbauer. Myeloid PTEN deficiency impairs tumor immune surveillance via immune checkpoint inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 527.

We here establish a mouse cancer model called Multi-Hit that allows for the evaluation of oncogene cooperativities in tumor development. The model is based on the stochastic expression of oncogene combinations ('hits') that are mediated by Cre in a given tissue. Cells with cooperating hits are positively selected and give rise to tumors. We used this approach to evaluate the requirement of Ras downstream effector pathways in tumorigenesis.

Inflammation is a strong promoter of colorectal cancer formation. Colorectal tumor cells establish heterotypic interactions with inflammatory cells in the stroma that are important for tumor angiogenesis and invasiveness. Recent studies in genetically modified mice have identified transcription factors and signaling networks that are implicated in these heterotypic tumor-stroma interactions and modulate preconditions of tumor formation such as chronic inflammation. Here, tumor-promoting and tumor-adverse effects of cytokine-activated transcription factors STAT1 and STAT3 in inflammatory cells and colorectal cancer cells are discussed.

The choice of an expression vector is a critical step in the field of recombinant protein production in mammalian cells lines. Most expression vectors used in the field are sensitive to the surrounding chromatin to their integration site into the host genome cell. This so-called chromatin positional effects influences the expression levels of the transgene and tends to silence its expression over time. Bacterial artificial chromosomes (BACs) are vectors that can accommodate inserts of up to 400 kb. Due to the large cloning capacity, BACs can harbour an entire locus with all or most of the regulatory elements controlling the expression of a gene. Therefore, BACs contain their own natural chromatin domain and are subjected to chromatin positional effects to a lesser extend or not at all. This makes cell lines generated with BAC-based expression vectors more predictable in terms of protein production and stability. In this chapter, we explore the use of BACs as expression vectors for recombinant protein production in mammalian cells.

The choice of an expression vector is a critical step in the field of recombinant protein production in mammalian cells lines. Most expression vectors used in the field are sensitive to the surrounding chromatin to their integration site into the host genome cell. This so-called chromatin positional effects influences the expression levels of the transgene and tends to silence its expression over time. Bacterial artificial chromosomes (BACs) are vectors that can accommodate inserts of up to 400 kb. Due to the large cloning capacity, BACs can harbour an entire locus with all or most of the regulatory elements controlling the expression of a gene. Therefore, BACs contain their own natural chromatin domain and are subjected to chromatin positional effects to a lesser extend or not at all. This makes cell lines generated with BAC-based expression vectors more predictable in terms of protein production and stability. In this chapter, we explore the use of BACs as expression vectors for recombinant protein production in mammalian cells.

Chronic liver injury, liver fibrosis and formation of hepatocellular carcinoma are intimately linked and represent a major medical challenge since treatment options are limited. Therefore, it is important to identify cellular and molecular pathways that promote liver damage or provide hepatoprotection for development of therapeutic approaches. Recently, the transcription factors STAT3 and STAT5 have been implicated in liver fibrosis induced by cholestatic liver damage. In this review, we summarize our current knowledge about STAT proteins in liver fibrosis and focus on common activities that underlie the hepatoprotective mechanisms regulated by IL-6/gp130/STAT3 and GH/STAT5/IGF-1 signaling pathways.