Bailu Peng

KrasG12D-Induced IKK2/β/NF-κB Activation by IL-1α and p62 Feedforward Loops Is Required for Development of Pancreatic Ductal Adenocarcinoma

Constitutive Kras and NF-κB activation is identified as signature alterations in pancreatic ductal adenocarcinoma (PDAC). However, how NF-κB is activated in PDAC is not yet understood. Here, we report that pancreas-targeted IKK2/β inactivation inhibited NF-κB activation and PDAC development in Kras(G12D) and Kras(G12D);Ink4a/Arf(F/F) mice, demonstrating a mechanistic link between IKK2/β and Kras(G12D) in PDAC inception. Our findings reveal that Kras(G12D)-activated AP-1 induces IL-1α, which, in turn, activates NF-κB and its target genes IL-1α and p62, to initiate IL-1α/p62 feedforward loops for inducing and sustaining NF-κB activity. Furthermore, IL-1α overexpression correlates with Kras mutation, NF-κB activity, and poor survival in PDAC patients. Therefore, our findings demonstrate the mechanism by which IKK2/β/NF-κB is activated by Kras(G12D) through dual feedforward loops of IL-1α/p62.

NF-κB and AP-1 Connection: Mechanism of NF-κB-Dependent Regulation of AP-1 Activity

ABSTRACT Nuclear factor κB (NF-κB) and activator protein 1 (AP-1) transcription factors regulate many important biological and pathological processes. Activation of NF-κB is regulated by the inducible phosphorylation of NF-κB inhibitor IκB by IκB kinase. In contrast, Fos, a key component of AP-1, is primarily transcriptionally regulated by serum responsive factors (SRFs) and ternary complex factors (TCFs). Despite these different regulatory mechanisms, there is an intriguing possibility that NF-κB and AP-1 may modulate each other, thus expanding the scope of these two rapidly inducible transcription factors. To determine whether NF-κB activity is involved in the regulation of fos expression in response to various stimuli, we analyzed activity of AP-1 and expression of fos, fosB, fra-1, fra-2, jun, junB, and junD, as well as AP-1 downstream target gene VEGF, using MDAPanc-28 and MDAPanc-28/IκBαM pancreatic tumor cells and wild-type, IKK1−/−, and IKK2−/− murine embryonic fibroblast cells. Our results show that elk-1, a member of TCFs, is one of the NF-κB downstream target genes. Inhibition of NF-κB activity greatly decreased expression of elk-1. Consequently, the reduced level of activated Elk-1 protein by extracellular signal-regulated kinase impeded constitutive, serum-, and superoxide-inducible c-fos expression. Thus, our study revealed a distinct and essential role of NF-κB in participating in the regulation of elk-1, c-fos, and VEGF expression.

The function of multiple IκB: NF-κB complexes in the resistance of cancer cells to Taxol-induced apoptosis

The Rel/NF-κB transcription factors play a key role in the regulation of apoptosis and in tumorigenesis by controlling the expressions of specific genes. To determine the role of the constitutive activity of RelA in tumorigenesis, we generated pancreatic tumor cell lines that express a dominant negative mutant of IκBα (IκBαM). In this report, we show that the inhibition of constitutive NF-κB activity, either by ectopic expression of IκBαM or by treating the cells with a proteasome inhibitor PS-341 which blocks intracellular degradation of IκBα proteins, downregulates the expression of bcl-xl. We identified two putative NF-κB binding sites (κB/A and B) in the bcl-xl promoter and found that these two sites interact with different NF-κB proteins. p65/p50 heterodimer interacts with κB/A site whereas p50/p50 homodimer interacts with κB/B. The bcl-xl promoter reporter gene assays reveal that NF-κB dependent transcriptional activation is mainly mediated by κB/A site, indicating that bcl-xl is one of the downstream target genes regulated by RelA/p50. Both IκBαM and PS-341 completely abolish NF-κB DNA binding activity; however, PS-341, but not ectopic expression of IκBαM, sensitized cells to apoptosis induced by Taxol. This is due to the Taxol-mediated reactivation of RelA through phosphorylation and degradation of IκBβ and the re-expression of NF-κB regulated bcl-xl gene in these cancer cells as ectopic expression of the bcl-xl gene confers resistance to Taxol-induced apoptosis in PS-341 sensitized cells. These results demonstrate the important function of various NF-κB/IκB complexes in regulating anti-apoptotic genes in response to apoptotic stimuli, and they raise the possibility that NF-κB : IκBα and NF-κB : IκBβ complexes are regulated by different upstream activators, and that NF-κB plays a key role in pancreatic tumorigenesis.

Mechanisms of Proinflammatory Cytokine-Induced Biphasic NF-κB Activation

Abstract The transcription factor NF-κB regulates genes involved in innate and adaptive immune response, inflammation, apoptosis, and oncogenesis. Proinflammatory cytokines induce the activation of NF-κB in both transient and persistent phases. We investigated the mechanism for this biphasic NF-κB activation. Our results show that MEKK3 is essential in the regulation of rapid activation of NF-κB, whereas MEKK2 is important in controlling the delayed activation of NF-κB in response to stimulation with the cytokines TNF-α and IL-1α. MEKK3 is involved in the formation of the IκBα:NF-κB/IKK complex, whereas MEKK2 participates in assembling the IκBβ:NF-κB/IKK complex; these two distinct complexes regulate the proinflammatory cytokine-induced biphasic NF-κB activation. Thus, our study reveals a novel mechanism in which different MAP3K and IκB isoforms are involved in specific complex formation with IKK and NF-κB for regulating the biphasic NF-κB activation. These findings provide further insight into the regulation of cytokine-induced specific and temporal gene expression.

Regulation of Nuclear Translocation of HDAC3 by IκBα Is Required for Tumor Necrosis Factor Inhibition of Peroxisome Proliferator-activated Receptor γ Function

Inhibition of peroxisome proliferator-activated receptor γ (PPARγ) function by TNF-α contributes to glucose and fatty acid metabolic disorders in inflammation and cancer, although the molecular mechanism is not fully understood. In this study, we demonstrate that nuclear translocation of HDAC3 is regulated by TNF-α, and this event is required for inhibition of transcriptional activity of PPARγ by TNF-α. HDAC3 is associated with IκBα in the cytoplasm. After IκBα degradation in response to TNF-α, HDAC3 is subject to nuclear translocation, leading to an increase in HDAC3 activity in the nucleus. This event leads to subcellular redistribution of HDAC3. Knock-out of IκBα, but not p65 or p50, leads to disappearance of HDAC3 in the cytoplasm, which is associated with HDAC3 enrichment in the nucleus. These data suggest that inhibition of PPARγ by TNF-α is not associated with a reduction in the DNA binding activity of PPARγ. Rather, these results suggest that IκBα-dependent nuclear translocation of HDAC3 is responsible for PPARγ inhibition by TNF-α.

Keratinocyte Growth Factor/Fibroblast Growth Factor-7-regulated Cell Migration and Invasion through Activation of NF-κB Transcription Factors

Keratinocyte growth factor (KGF)/fibroblast growth factor-7 (FGF-7) is a paracrine- and epithelium-specific growth factor produced by cells of mesenchymal origin. It acts exclusively through FGF-7 receptor (FGFR2/IIIb), which is expressed predominantly by epithelial cells, but not by fibroblasts, suggesting that it might function as a paracrine mediator of mesenchymal-epithelial interactions. KGF/FGF-7 plays an essential role in the growth of epithelial cells and is frequently overexpressed in cancers of epithelial origin such as pancreatic cancer, switching paracrine stimulation of KGF/FGF-7 to an autocrine loop. Less is known, however, about the signaling pathways by which KGF/FGF-7 regulates the response of epithelial cells. To delineate the signaling pathways activated by KGF/FGF-7 and examine cellular response to KGF/FGF-7 stimulation, we performed functional analysis of KGF/FGF-7 action. In this report, we show that KGF/FGF-7 activated nuclear factor κB (NF-κB), which in turn induced expression of VEGF, MMP-9, and urokinase-type plasminogen activator and increased migration and invasion of KGF/FGF-7-stimulated human pancreatic ductal epithelial cells. Expression of phosphorylation-defective IκBα (IκBαS32A,S36A), which blocked NF-κB activation, inhibited KGF/FGF-7-induced gene expression and cell migration and invasion. Our results demonstrate for the first time that KGF/FGF-7 induces NF-κB activation and that NF-κB plays an essential role in regulation of KGF/FGF-7-inducible gene expression and KGF/FGF-7-initiated cellular responses. Thus, these findings identify one signaling pathway for KGF/FGF-7-regulated cell migration and invasion and suggest that paracrine sources of KGF/FGF-7 are one of the malignancy-contributing factors from tumor stroma.

Overexpression of synuclein-γ in pancreatic adenocarcinoma

Currently, pancreatic adenocarcinoma is the fourth leading cause of cancer‐related death in the United States. Despite the advances in pancreatic carcinoma research, patients with this devastating disease have a very poor prognosis. To identify the gene expression profile of pancreatic carcinoma, an important step in the process of developing new diagnostic and therapeutic strategies, the authors investigated the alteration of gene expression in this disease.

Secreted Interleukin-1α Induces a Metastatic Phenotype in Pancreatic Cancer by Sustaining a Constitutive Activation of Nuclear Factor-κB

Transcription factor nuclear factor-κB (NF-κB) is constitutively activated in most pancreatic cancer tissues and cell lines but not in normal pancreas nor in immortalized/nontumorigenic human pancreatic ductal epithelial cells. Inhibition of constitutive NF-κB activation in pancreatic cancer cell lines suppresses tumorigenesis and tumor metastasis. Recently, we identified autocrine secretion of proinflammatory cytokine interleukin (IL)-1α as the mechanism of constitutive NF-κB activation in metastatic pancreatic cancer cell lines. However, the role of IL-1α in determining the metastatic potential of pancreatic tumor remains to be further investigated. In the current study, we stably expressed IL-1α in the nonmetastatic, IL-1α–negative MiaPaCa-2 cell lines. Our results showed that the secretion of IL-1α in MiaPaCa-2 cells constitutively activated NF-κB and increased the expression of NF-κB downstream genes involved in the different steps of the metastatic cascade, such as urokinase-type plasminogen activator, vascular endothelial growth factor, and IL-8. MiaPaCa-2/IL-1α cells showed an enhanced cell invasion in vitro compared with parental MiaPaCa-2 cells and induced liver metastasis in an orthotopic mouse model. The metastatic phenotype induced by IL-1α was inhibited by the expression of phosphorylation-defective IκB (IκB S32, 36A), which blocked NF-κB activation. Consistently, silencing the expression of IL-1α by short hairpin RNA in the highly metastatic L3.6pl pancreatic cancer cells completely suppressed their metastatic spread. In summary, these findings showed that IL-1α plays key roles in pancreatic cancer metastatic behavior through the constitutive activation of NF-κB. Our findings further support the possible link between inflammation and cancer and suggest that IL-1α may be a potential therapeutic target for treating pancreatic adenocarcinoma. (Mol Cancer Res 2009;7(5):624–33)

Function of Polo-like Kinase 3 in NF-κB-mediated Proapoptotic Response

RelA, the p65 subunit of NF-κB transcription factors, plays a key role in regulation of antiapoptotic and proapoptotic responses. However, the downstream target genes regulated by RelA-NF-κB in the initiation of proapoptotic signaling were not identified. We previously showed that RelA-NF-κB functioned as a proapoptotic factor by activating the p53-signaling pathway in response to doxycycline-induced superoxide. In the present study, we demonstrate that the ability of doxycycline/superoxide to induce expression of polo-like kinase 3 (Plk3) depends on NF-κB activity. We identified a κB binding site in the promoter of Plk3, and this κB site is directly involved in its induction by the RelA-NF-κB complex. Plk3 formed a complex with p53 and was involved in the phosphorylation of p53 on Ser-20 in response to superoxide. Inhibition of Plk3 expression by Plk3 small interfering RNA suppressed the doxycycline/superoxide-mediated apoptosis. Overexpression of wild-type Plk3 in HCT116 p53+/+ cells induced rapid apoptosis, whereas overexpression of wild-type Plk3 in HCT116 p53–/– cells and the kinase-defective mutant Plk3K91R in p53+/+ cells induced delayed onset of apoptosis. Furthermore, mutagenesis of Plk3 showed that the N-terminal domain (amino acids 1–26) is essential for the induction of delay onset of apoptosis. These data show that Plk3 is a RelA-NF-κB-regulated gene that induces apoptosis in both p53-dependent and -independent signaling pathways, suggesting a possible mechanism for RelA-NF-κB-regulated proapoptotic responses.

Defective feedback regulation of NF-κB underlies Sjögren’s syndrome in mice with mutated κB enhancers of the IκBα promoter

Feedback regulation of transcription factor NF-κB by its inhibitor IκBα plays an essential role in control of NF-κB activity. To understand the biological significance of IκBα-mediated feedback regulation of NF-κB, we generated mice harboring mutated κB enhancers in the promoter of the IκBα gene (IκBαM/M) to inhibit NF-κB–regulated IκBα expression. Here, we report that these mutant mice are defective in NF-κB–induced expression of IκBα. This defective feedback regulation of NF-κB by IκBα not only altered activity of NF-κB, but also the expression of NF-κB–regulated genes. As a result, IκBαM/M, the homozygous knock-in mice with mutated κB enhancers in the IκBα promoter, acquire shorten life span, hypersensitivity to septic shock, abnormal T-cell development and activation, and Sjögren’s Syndrome. These findings therefore demonstrate that the IκBα-mediated feedback regulation of NF-κB has an essential role in controlling T-cell development and functions, provide mechanistic insight into the development of Sjögren’s Syndrome, and suggest the potential of NF-κB signaling as a therapeutic target for Sjögren’s Syndrome and other autoimmune diseases.