Christoph K. Weber

Mitogenic and antiapoptotic role of constitutive NF‐κB/Rel activity in pancreatic cancer

The transcription factor NF‐κB/Rel was found to be constitutively activated in human pancreatic cancer. RelA is present in the nucleus in primary human pancreatic cancer samples as well as in pancreatic cancer cell lines. NF‐κB/Rel–binding activity consists of NF‐κB1(p50) and RelA(p65). Constitutive NF‐κB/Rel activity correlates with IκB kinase (IKK) activity and can be blocked by dominant negative mutants of IKKβ and to a lesser extent by IKKα. Constitutive NF‐κB/Rel activity and the transactivation potential of RelA(p65) can be inhibited by dominant negative mutant Ras, the PI3 kinase inhibitor LY294002, or dominant negative mutant Akt kinase. Transfection of a dominant negative mutant epidermal growth factor receptor (EGF‐R), EGF‐R kinase inhibitor Tyrphostin and LY 294002 blocked IKK activity and NF‐κB–dependent transcription. Inhibition of constitutive IKK or NF‐κB/Rel activity increased the number of apoptotic cells. Stably expressing a nondegradable form of IκBα inhibited anchorage‐dependent and ‐independent proliferation in MiaPaCa2 and Panc1 cells. Our data demonstrate that an EGF‐R/Ras/PI3 kinase/Akt/IKK‐dependent pathway contributes to constitutive NF‐κB/Rel activity in pancreatic cancer. Inhibition of NF‐κB/Rel activity reveals a mitogenic and antiapoptotic role for NF‐κB/Rel in pancreatic cancer.

Sulindac sulfide inhibits Ras signaling.

The non-steroidal anti-inflammatory drug sulindac is used in cancer prevention and therapy, but the molecular aspects of its anti-tumor effect remain unresolved. In vivo the prodrug sulindac, is converted into the metabolite sulindac sulfide. We found that sulindac sulfide strongly inhibits Ras induced malignant transformation and Ras/Raf dependent transactivation. Sulindac sulfide decreases the Ras induced activation of its main effector, the c-Raf-1 kinase. In vitro sulindac sulfide directly binds to the Ras gene product p21ras in a non-covalent manner. Moreover, we can show that sulindac sulfide inhibits the interaction of p21ras with the p21ras binding domain of the Raf protein. In addition, sulindac sulfide can impair the nucleotide exchange on p21ras by CDC25 as well as the acceleration of the p21ras GTPase reaction by p120GAP. Due to its action at the most critical site in Ras signaling we propose sulindac sulfide as a lead compound in the search for novel anti-cancer drugs which directly inhibit Ras mediated cell proliferation and malignant transformation.

Constitutive IKK2 activation in acinar cells is sufficient to induce pancreatitis in vivo

Activation of the inhibitor of NF-kappaB kinase/NF-kappaB (IKK/NF-kappaB) system and expression of proinflammatory mediators are major events in acute pancreatitis. However, the in vivo consequences of IKK activation on the onset and progression of acute pancreatitis remain unclear. Therefore, we modulated IKK activity conditionally in pancreatic acinar cells. Transgenic mice expressing the reverse tetracycline-responsive transactivator (rtTA) gene under the control of the rat elastase promoter were generated to mediate acinar cell-specific expression of IKK2 alleles. Expression of dominant-negative IKK2 ameliorated cerulein-induced pancreatitis but did not affect activation of trypsin, an initial event in experimental pancreatitis. Notably, expression of constitutively active IKK2 was sufficient to induce acute pancreatitis. This acinar cell-specific phenotype included edema, cellular infiltrates, necrosis, and elevation of serum lipase levels as well as pancreatic fibrosis. IKK2 activation caused increased expression of known NF-kappaB target genes, including mediators of the inflammatory response such as TNF-alpha and ICAM-1. Indeed, inhibition of TNF-alpha activity identified this cytokine as an important effector of IKK2-induced pancreatitis. Our data identify the IKK/NF-kappaB pathway in acinar cells as being key to the development of experimental pancreatitis and the major factor in the inflammatory response typical of this disease.

From acinar cell damage to systemic inflammatory response: current concepts in pancreatitis.

Acute pancreatitis represents a local inflammatory disorder with severe systemic consequences. Significant progress in understanding the pathophysiology of acute pancreatitis has been achieved in recent years. However, there is no clear concept about initialization and propagation of the disease both in experimental models and in humans. Furthermore, reliable strategies to evaluate prognosis and perform therapy are still missing. The review focuses on mechanisms originating from acinar cells leading to a systemic inflammatory response in experimental pancreatitis.

Protein kinase D2 mediates activation of nuclear factor κB by Bcr-Abl in Bcr-Abl+ human myeloid leukemia cells

The Bcr-Abl tyrosine kinase activates various signaling pathways including nuclear factor κB that mediate proliferation, transformation, and apoptosis resistance in Bcr-Abl+ myeloid leukemia cells. Here we report that protein kinase (PK) D2, a serine threonine kinase of the PKD family, is a novel substrate of Bcr-Abl. PKD2 was found to be the major isoform of the PKD family expressed in chronic myeloid leukemia cells and is tyrosine phosphorylated by Bcr-Abl in its pleckstrin homology domain. A mutant that mimicks tyrosine phosphorylation of PKD2 in the pleckstrin homology domain activates nuclear factor κB independently of its catalytic activity. Furthermore, our data show that Bcr-Abl–induced activation of the nuclear factor κB cascade in LAMA84 cells is largely mediated by tyrosine-phosphorylated PKD2. These data present a novel mechanism of Bcr-Abl–induced nuclear factor κB activation in myeloid leukemia. Targeting PKD2 tyrosine phosphorylation, not its kinase activity, could be a novel therapeutic approach for the treatment of Bcr-Abl+ myeloid leukemia.

Activity of Rap1 Is Regulated by Bombesin, Cell Adhesion, and Cell Density in NIH3T3 Fibroblasts

Rap1 and Ras are homologous GTPases that are implicated in cell proliferation and differentiation. At present, little is known about the regulation of Rap1 activity. Using a recently developed assay with activation-specific probes, we found increased activity of endogenous Rap1 in NIH3T3 cells after stimulation with the neuropeptide growth factor bombesin in a concentration- and time-dependent manner. The activity of endogenous Ras was unaffected. Analysis of putative effectors showed no activation of c-Raf-1 or B-Raf after bombesin stimulation. However, MAPK/Erk-phosphorylation and the proliferation rate was increased. In addition, Rap1 was activated during cell adhesion to coated and uncoated tissue culture plates, as well as in response to various mitogens. Surprisingly, the basal Rap1 activity was observed to be cell density-dependent, with low levels when cells were reaching confluency. The results suggest that Rap1 acts as an important mediator of mitogenic signals distinct to Ras activation.

The GABP-responsive Element of the Interleukin-2 Enhancer Is Regulated by JNK/SAPK-activating Pathways in T Lymphocytes

T cell activation leads via multiple intracellular signaling pathways to rapid induction of interleukin-2 (IL-2) expression, which can be mimicked by costimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) and ionomycin. We have identified a distal IL-2 enhancer regulated by the Raf-MEK-ERK signaling pathway, which can be induced by TPA/ionomycin treatment. It contains a dyad symmetry element (DSE) controlled by the Ets-like transcription factor GA-binding protein (GABP), a target of activated ERK. TPA/ionomycin treatment of T cells stimulates both mitogen-activated ERK, as well as the stress-activated mitogen-activated protein kinase family members JNK/SAPK and p38. In this study, we investigated the contribution of the stress-activated pathways to the induction of the distal IL-2 enhancer. We show that JNK- but not p38-activating pathways regulate the DSE activity. Furthermore, the JNK/SAPK signaling pathway cooperates with the Raf-MEK-ERK cascade in TPA/ionomycin-induced DSE activity. In T cells, overexpression of SPRK/MLK3, an activator of JNK/SAPK, strongly induces DSE-dependent transcription and dominant negative kinases of SEK and SAPK impair TPA/ionomycin-induced DSE activity. Blocking both ERK and JNK/SAPK pathways abolishes the DSE induction. The inducibility of the DSE is strongly dependent on the Ets-core motifs, which are bound by GABP. Both subunits of GABP are phosphorylated upon JNK activation in vivo and three different isoforms of JNK/SAPK, but not p38, in vitro. Our data suggest that GABP is targeted by signaling events from both ERK and JNK/SAPK pathways. GABP therefore is a candidate for signal integration and regulation of IL-2 transcription in T lymphocytes.

Mitogenic signaling of Ras is regulated by differential interaction with Raf isozymes

In the mitogenic signaling cascade interaction of Ras with Raf represents a critical step for the regulation of cell growth and differentiation. The major effector of Ras, the serine/threonine kinase Raf exists as three isoforms with different tissue distributions. We demonstrate that transient transfection of oncogenic Ha-Ras leads to a preferential activation of endogenous c-Raf-1 in HEK 293 cells as opposed to A-Raf. In vitro binding studies using purified Ras binding domains of Raf as well as in vivo bindings tests with full length molecules reveals significantly lower binding affinities of A-Raf to Ha-Ras as compared to other Raf isoforms. The Ras-binding interface of c-Raf differs from A-Raf by a conservative Arg to Lys exchange at residue 59 or 22 respectively. Mutational analysis reveals that this residue represents a point of isozyme discrimination: c-Raf-R59K binds Ha-Ras weaker than the wildtype, likewise A-Raf-K22R increases its affinity to Ha-Ras in vivo and in vitro. Differential binding affinities are reflected in downstream signaling. Immunecomplex kinase assays reveal that Ha-Ras mediated Raf activation is decreased for c-Raf-R59K and increased for A-Raf-K22R when compared to the respective wildtype forms. Thus our observations introduce a new level of isoform discrimination in Ras/Raf signaling as a functional consequence of a conservative amino acid exchange in the Ras binding domains.

TGFα Transgenic Mice

Pancreatic cancer is a devastating disease with a fatal prognosis due to late diagnosis and resistance to radiation and chemotherapy. The average survival after diagnosis is still 3 to 8 months. In the last few years genetic alterations in cancer-causing genes have been identified in tumors and putative premalignant lesions using microdissection techniques. However, the functional consequence of these genetic alterations for pancreatic growth and differentiation is unknown. TGFα overexpressed in the pancreas causes the development of tubular structures and fibrosis. Mice older than one year develop ductal pancreatic cancer. Crossbreeding these mice with p53 knockout mice dramatically accelerated tumor development. Moveover, tumors developing in these mice show frequently biallelic deletion of the Ink4a locus or LOH of SMAD4. These mice represent the first model of pancreatic adenocarcinomas with genetic alterations as well as growth characteristics similar to the human disease.

The RafC1 Cysteine-Rich Domain Contains Multiple Distinct Regulatory Epitopes Which Control Ras-Dependent Raf Activation

ABSTRACT Activation of c-Raf-1 (referred to as Raf) by Ras is a pivotal step in mitogenic signaling. Raf activation is initiated by binding of Ras to the regulatory N terminus of Raf. While Ras binding to residues 51 to 131 is well understood, the role of the RafC1 cysteine-rich domain comprising residues 139 to 184 has remained elusive. To resolve the function of the RafC1 domain, we have performed an exhaustive surface scanning mutagenesis. In our study, we defined a high-resolution map of multiple distinct functional epitopes within RafC1 that are required for both negative control of the kinase and the positive function of the protein. Activating mutations in three different epitopes enhanced Ras-dependent Raf activation, while only some of these mutations markedly increased Raf basal activity. One contiguous inhibitory epitope consisting of S177, T182, and M183 clearly contributed to Ras-Raf binding energy and represents the putative Ras binding site of the RafC1 domain. The effects of all RafC1 mutations on Ras binding and Raf activation were independent of Ras lipid modification. The inhibitory mutation L160A is localized to a position analogous to the phorbol ester binding site in the protein kinase C C1 domain, suggesting a function in cofactor binding. Complete inhibition of Ras-dependent Raf activation was achieved by combining mutations K144A and L160A, which clearly demonstrates an absolute requirement for correct RafC1 function in Ras-dependent Raf activation.