Harald J. Maier

NF-κB promotes epithelial–mesenchymal transition, migration and invasion of pancreatic carcinoma cells

The transcription factor NF-kappaB is constitutively active in pancreatic adenocarcinoma. Here we explore the contribution of NF-kappaB to the malignant phenotype of pancreatic cancer cells in addition to its anti-apoptotic role. Block of NF-kappaB signalling by non-destructible IkappaBalpha rendered cells resistant to TGF-beta-induced epithelial-mesenchymal transition (EMT). In contrast, NF-kappaB activation by TNF-alpha or expression of constitutively active IKK2 induced an EMT-phenotype with up-regulation of vimentin and ZEB1, and down-regulation of E-cadherin. EMT could also be induced in cells with defective TGF-beta signalling. Functional assays demonstrated reduced or strongly enhanced migration and invasion upon NF-kappaB inhibition or activation, respectively.

FOXO1 is a tumor suppressor in classical Hodgkin lymphoma

The FOXO transcription factors control proliferation and apoptosis in different cell types. Their activity is regulated by posttranslational modifications, mainly by the PI3K-PKB pathway, which controls nuclear export and degradation. We show that FOXO1 is highly expressed in normal germinal center B cells as well as in non-Hodgkin lymphomas, including follicular lymphoma, diffuse large B-cell lymphoma, mucosa-associated lymphoid tissue non-Hodgkin lymphoma, B-cell chronic lymphocytic leukemia, and mantle cell lymphoma. In contrast, in 31 of 32 classical Hodgkin lymphoma (cHL) cases, Hodgkin and Reed-Sternberg cells were FOXO1 negative. Neoplastic cells of nodular lymphocyte-predominant Hodgkin lymphoma were negative in 14 of 20 cases. FOXO1 was down-regulated in cHL cell lines, whereas it was expressed in non-Hodgkin lymphoma cell lines at levels comparable with normal B cells. Ectopic expression of a constitutively active FOXO1 induced apoptosis in cHL cell lines and blocked proliferation, accompanied with cell-cycle arrest in the G(0)/G(1) phase. We found that, in cHL cell lines, FOXO1 is inactivated by multiple mechanisms, including constitutive activation of AKT/PKB and MAPK/ERK kinases and up-regulation of microRNAs miR-96, miR-182, and miR-183. These results suggest that FOXO1 repression contributes to cHL lymphomagenesis.

Cardiomyocyte-specific IκB kinase (IKK)/NF-κB activation induces reversible inflammatory cardiomyopathy and heart failure

Inflammation is a major factor in heart disease. IκB kinase (IKK) and its downstream target NF-κB are regulators of inflammation and are activated in cardiac disorders, but their precise contributions and targets are unclear. We analyzed IKK/NF-κB function in the heart by a gain-of-function approach, generating an inducible transgenic mouse model with cardiomyocyte-specific expression of constitutively active IKK2. In adult animals, IKK2 activation led to inflammatory dilated cardiomyopathy and heart failure. Transgenic hearts showed infiltration with CD11b+ cells, fibrosis, fetal reprogramming, and atrophy of myocytes with strong constitutively active IKK2 expression. Upon transgene inactivation, the disease was reversible even at an advanced stage. IKK-induced cardiomyopathy was dependent on NF-κB activation, as in vivo expression of IκBα superrepressor, an inhibitor of NF-κB, prevented the development of disease. Gene expression and proteomic analyses revealed enhanced expression of inflammatory cytokines, and an IFN type I signature with activation of the IFN-stimulated gene 15 (ISG15) pathway. In that respect, IKK-induced cardiomyopathy resembled Coxsackievirus-induced myocarditis, during which the NF-κB and ISG15 pathways were also activated. Vice versa, in cardiomyocytes lacking the regulatory subunit of IKK (IKKγ/NEMO), the induction of ISG15 was attenuated. We conclude that IKK/NF-κB activation in cardiomyocytes is sufficient to cause cardiomyopathy and heart failure by inducing an excessive inflammatory response and myocyte atrophy.

FoxO3 induces reversible cardiac atrophy and autophagy in a transgenic mouse model

AIMS The transcription factor FoxO3 contributes to anti-hypertrophic signalling in the heart presumably by regulating autophagic-lysosomal and ubiquitin-proteasomal pathways. We wanted to study FoxO3 function in the adult heart in vivo by expressing a constitutively active mutant of FoxO3 in transgenic mice. METHODS AND RESULTS We generated transgenic mice in which a tetracycline-regulated constitutively active FoxO3 transgene (FoxO3-CA) is controlled by the heart-specific α-myosin heavy chain promoter. Cardiac-specific expression in adult mice resulted in a decrease in heart weight by 25% and a reduction in stroke volume and cardiac output. The decrease in heart size was due to a reduction in the size of individual cardiomyocytes, whereas there was no evidence for increased cell death. FoxO3 activation was accompanied by the initiation of a foetal gene programme with increased expression of β-myosin heavy chain and natriuretic peptides, and by the activation of AKT and mammalian target of rapamycin signalling. As shown by electron microscopy, FoxO3-CA massively stimulated destruction of sarcomeres and autophagy, and induced expression of LC3-II and BNIP3. When FoxO3-CA expression was shut off in affected mice, cardiac atrophy and dysfunction as well as molecular markers were normalized within 1 month. FoxO3-CA expression did not counteract hypertrophy induced by transverse aortic constriction. CONCLUSION Heart-specific expression of constitutively active FoxO3 leads to reversible heart atrophy. The reversibility of the phenotype suggests a remarkable ability of the adult myocardium to respond to different regulatory cues.

Critical Role of RelB Serine 368 for Dimerization and p100 Stabilization

In mature B cells RelB-containing complexes are constitutively present in the nucleus, and they are less susceptible to inhibitory κB proteins. In most other cell types inhibitory κB proteins prevent nuclear translocation and activation of NFκB. We reasoned that this characteristic might be because of post-translational modifications of RelB. In Drosophila, signal-dependent phosphorylation of the Rel homologue Dorsal at serine 317 has been shown to be critical for nuclear import. The evolutionary conservation of this serine prompted us to analyze the function of the corresponding site in RelB. As a model system we used the murine S107 plasmacytoma cell line, which lacks endogenous RelB expression. Analysis of S107 cells expressing wild type RelB and serine 368 mutants reveals that serine 368 is not required for nuclear import but that it is critical for RelB dimerization with other members of the NFκB family. Similar effects were obtained when the conserved serine in RelA was mutated. We further demonstrate that expression of functional RelB, but not of serine 368 mutants, severely reduces p52 generation and strongly increases expression of the p52 precursor, p100. Wild type RelB, but not mutant RelB, prolonged p100 half-life. We therefore suggest an inhibitory effect of RelB on p100 processing, which is possibly regulated in a signal-dependent manner.

Epithelial-Mesenchymal Transition in Pancreatic Carcinoma

Pancreatic carcinoma is the fourth-leading cause of cancer death and is characterized by early invasion and metastasis. The developmental program of epithelial-mesenchymal transition (EMT) is of potential importance for this rapid tumor progression. During EMT, tumor cells lose their epithelial characteristics and gain properties of mesenchymal cells, such as enhanced motility and invasive features. This review will discuss recent findings pertinent to EMT in pancreatic carcinoma. Evidence for and molecular characteristics of EMT in pancreatic carcinoma will be outlined, as well as the connection of EMT to related topics, e.g., cancer stem cells and drug resistance.

FOXO1 repression contributes to block of plasma cell differentiation in classical Hodgkin Lymphoma

The survival of classical Hodgkin lymphoma (cHL) cells depends on activation of NF-κB, JAK/STAT, and IRF4. Whereas these factors typically induce the master regulator of plasma cell (PC) differentiation PRDM1/BLIMP-1, levels of PRDM1 remain low in cHL. FOXO1, playing a critical role in normal B-cell development, acts as a tumor suppressor in cHL, but has never been associated with induction of PC differentiation. Here we show that FOXO1 directly upregulates the full-length isoform PRDM1α in cHL cell lines. We also observed a positive correlation between FOXO1 and PRDM1 expression levels in primary Hodgkin-Reed-Sternberg cells. Further, we show that PRDM1α acts as a tumor suppressor in cHL at least partially by blocking MYC. Here we provide a link between FOXO1 repression and PRDM1α downregulation in cHL and identify PRDM1α as a tumor suppressor in cHL. The data support a potential role for FOXO transcription factors in normal PC differentiation.

Long-Term IKK2/NF-κB Signaling in Pancreatic β-Cells Induces Immune-Mediated Diabetes

Type 1 diabetes is a multifactorial inflammatory disease in genetically susceptible individuals characterized by progressive autoimmune destruction of pancreatic β-cells initiated by yet unknown factors. Although animal models of type 1 diabetes have substantially increased our understanding of disease pathogenesis, heterogeneity seen in human patients cannot be reflected by a single model and calls for additional models covering different aspects of human pathophysiology. Inhibitor of κB kinase (IKK)/nuclear factor-κB (NF-κB) signaling is a master regulator of inflammation; however, its role in diabetes pathogenesis is controversially discussed by studies using different inhibition approaches. To investigate the potential diabetogenic effects of NF-κB in β-cells, we generated a gain-of-function model allowing conditional IKK2/NF-κB activation in β-cells. A transgenic mouse model that expresses a constitutively active mutant of human IKK2 dependent on Pdx-1 promoter activity (IKK2-CAPdx-1) spontaneously develops full-blown immune-mediated diabetes with insulitis, hyperglycemia, and hypoinsulinemia. Disease development involves a gene expression program mimicking virus-induced diabetes and allergic inflammatory responses as well as increased major histocompatibility complex class I/II expression by β-cells that could collectively promote diabetes development. Potential novel diabetes candidate genes were also identified. Interestingly, animals successfully recovered from diabetes upon transgene inactivation. Our data give the first direct evidence that β-cell–specific IKK2/NF-κB activation is a potential trigger of immune-mediated diabetes. Moreover, IKK2-CAPdx-1 mice provide a novel tool for studying critical checkpoints in diabetes pathogenesis and mechanisms governing β-cell degeneration/regeneration.

Requirement of NEMO/IKKγ for effective expansion of KRAS-induced precancerous lesions in the pancreas

Pancreatic carcinoma, a leading cause of cancer death, is thought to develop out of pancreatic intraepithelial neoplasia (PanIN). PanIN lesions have not yet attained the fully malignant phenotype, but show increased proliferation and dysplasia, and frequently bear an oncogenic KRAS mutation. Pancreatic cancer development is associated with increased activity of the transcription factor NF-κB. NEMO (IKKγ) is a subunit of the IKK complex essential for the activation of canonical NF-κB signaling and has been ascribed both oncogenic and tumor-suppressive roles in gastrointestinal tumors. Here, we wanted to address the function of NEMO in pancreatic tumorigenesis. We therefore conditionally ablated NEMO in a mouse model for pancreatic carcinoma based on the expression of oncogenic KRAS in pancreatic precursor cells. Mice were analyzed for PanIN lesions and for the activation of associated signaling pathways. NEMO ablation in the pancreas, while in itself not causing any overt pathology, led to a drastic (>93%) decrease in the prevalence of both low-grade and high-grade PanIN in 10-month-old mice expressing oncogenic KRAS. Also, the inflammatory and fibrotic response associated with KRAS action in the pancreas was virtually abolished, including expression of inflammatory cytokines and activation of the interleukin-6/STAT3 axis. Moreover, the activation of MAPK signaling, Notch and KLF4 signaling normally observed in KRAS-induced PanIN was strongly reduced or absent when NEMO was ablated. Our study suggests that NEMO, an IKK subunit necessary for canonical NF-κB activation, is dispensable for normal pancreatic development and function, but essential for the propagation of KRAS-induced PanIN lesions.

BI 5700, a Selective Chemical Inhibitor of IκB Kinase 2, Specifically Suppresses Epithelial-Mesenchymal Transition and Metastasis in Mouse Models of Tumor Progression

Increasing evidence suggests that processes termed epithelial-mesenchymal transitions (EMTs) play a key role in therapeutic resistance, tumor recurrence, and metastatic progression. NF-κB signaling has been previously identified as an important pathway in the regulation of EMT in a mouse model of tumor progression. However, it remains unclear whether there is a broad requirement for this pathway to govern EMT and what the relative contribution of IKK family members acting as upstream NF-κB activators is toward promoting EMT and metastasis. To address this question, we have used a novel, small-molecule inhibitor of IκB kinase 2 (IKK2/IKKβ), termed BI 5700. We investigated the role of IKK2 in a number of mouse models of EMT, including TGFβ-induced EMT in the mammary epithelial cell line EpRas, CT26 colon carcinoma cells, and 4T1 mammary carcinoma cells. The latter model was also used to evaluate in vivo activities of BI 5700.We found that BI 5700 inhibits IKK2 with an IC(50) of 9 nM and was highly selective as compared to other IKK family members (IKK1, IKKε, and TBK1) and other kinases. BI 5700 effectively blocks NF-κB activity in EpRas cells and prevents TGFβ-induced EMT. In addition, BI 5700 reverts EMT in mesenchymal CT26 cells and prevents EMT in the 4T1 model. Oral application of BI 5700 significantly interferes with metastasis after mammary fat-pad injection of 4T1 cells, yielding fewer, smaller, and more differentiated metastases as compared to vehicle-treated control animals. We conclude that IKK2 is a key regulator of both the induction and maintenance of EMT in a panel of mouse tumor progression models and that the IKK2 inhibitor BI 5700 constitutes a promising candidate for the treatment of metastatic cancers.