SUMMARY 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 KrasG12D and KrasG12D;Ink4a/ArfF/F mice, demonstrating a mechanistic link between IKK2/β and KrasG12D in PDAC inception. Our findings reveal that KrasG12D-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 KrasG12D through dual feedforward loops of IL-1α/p62.

Nuclear factor B (NF-B) and activator protein 1 (AP-1) are key transcription factors that orchestrate expression of many genes involved in inflammation, embryonic development, lymphoid differentiation, oncogenesis, and apoptosis (48, 62). NF-B and AP-1 activities are induced by a plethora of physiological and environmental stimuli (5, 51).The activity of NF-B is regulated by its interaction with the family of NF-B inhibitors known as IB, which results in the formation of inactive NF-B-IB complexes in the cytoplasm (3,4,60). In response to various stimuli, the IB kinase complex (IKK) then phosphorylates the IB bound to the NF-B complexes as substrates (8,36,45,47). The subsequent proteasome-mediated degradation of IB exposes the nuclear localization signal (NLS) of NF-B, which releases the NF-B proteins to be translocated to the nucleus, where they regulate the transcription of specific genes (5, 48).AP-1 is a group of basic leucine zipper (bZIP) transcription factors consisting of the Fos (c-Fos, FosB, Fra1, and Fra2) and Jun (c-Jun, JunB, and JunD) families (54, 62). The predominant forms of AP-1 in most cells are Fos/Jun heterodimers which have a high affinity for binding to an AP-1 site, whereas Jun/Jun homodimers bind to the AP-1 site with low affinity (54,62). A number of studies have shown that serum and growth factors that induce AP-1 do so by activating the extracellular signal-regulated kinase (ERK) subgroup of mitogen-activated protein kinases (MAPKs) (9, 27, 55). These activated members of MAPKs translocate to the nucleus to phosphorylate and thereby transcriptionally activate a subfamily of ETS domain transcription factors known as ternary complex factors (TCFs) that bind to fos promoters (9,27,55,65,80). fos, fosB, and other members of the AP-1 family of transcription factors are mainly regulated at their transcription through serum responsive elements (SREs) in their promoters (57,76). For example, the regulation of c-fos expression is controlled by Elk, a member of TCFs that associates with the serum response factor (SRF) (11,28,49). The elk-1 gene encodes two spliced variants: elk-1 and an alternatively spliced variant known as ⌬elk-1, which is missing the SRF interaction domain and part of the elk-1 DNA binding domain (61). The ⌬Elk-1protein cannot form an SRF-dependent ternary complex with SRE to activate fos transcription (61). However, a variety of experiments have shown that Elk-1 proteins play a central role in the response of cells to many extracellular signals and control the expression of genes involved in cell cycle progression, differentiation, and apoptosis (62, 75). The mechanism by which Elk-1 activates transcription in response to various stimuli has been extensively studied; however, less is known about the regulation of elk-1 gene expression itself.Even though NF-B and AP-1 transcription factors are regulated by different mechanisms, they appear to be activated simultaneously by the same multitude of stimuli (1,19,37,43,71,78). A number of reports also showed that these transcrip...

Expression of embryonic stem cells (ESCs) markers (SOX2, OCT4, Nanog and Nestin) is crucial for progression of various human malignancies. The purpose of this study was to investigate the expression and prognostic impact of these molecules in nasopharyngeal carcinoma (NPC) patients by immunohistochemistry and immunofluorescence. In the present study, we found that the expression levels of SOX2, OCT4 and Nanog were highly expressed in NPC compared with the non-tumorous tissues. Furthermore, these proteins correlated significantly with several clinicalpathological factors and epithelial-mesenchymal transition (EMT)-associated indicators (E-cadherin/N-cadherin and Snail). In multivariate analyses, high expression of OCT4 (P = 0.013) and Nanog (P = 0.040), but not that of SOX2, was associated with worse survival and had strongly independent prognostic effects. Of note, OCT4 and Nanog were more frequently located at the invasive front of tumors, and correlated significantly with various aggressive behaviors including T classification, N classification, M classification and clinical stage. Furthermore, patients with co-expression of OCT4 and Nanog in the invasive front had significantly worse survival (P = 0.005). Interestingly, at the invasive front, these molecules correlated significantly with Nestin expression in endothelial cells (P<0.001). These findings provide evidence that ESCs biomarkers OCT4 and Nanog serves as independent prognostic factors for NPC. Additionally, cancer cells in the invasive front of NPC acquiring ESCs-like features should be maintained by vascular niches.

Inhibition of peroxisome proliferator-activated receptor ␥ (PPAR␥) function by TNF-␣ 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-␣, and this event is required for inhibition of transcriptional activity of PPAR␥ by TNF-␣. HDAC3 is associated with IB␣ in the cytoplasm. After IB␣ degradation in response to TNF-␣, 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 IB␣, 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 PPAR␥ by TNF-␣ is not associated with a reduction in the DNA binding activity of PPAR␥. Rather, these results suggest that IB␣-dependent nuclear translocation of HDAC3 is responsible for PPAR␥ inhibition by TNF-␣.PPAR␥ is a nuclear receptor in the family of peroxisome proliferatoractivated receptor (PPAR) 2 that includes PPAR␣, PPAR␥, and PPAR␦ (PPAR␤) (reviewed in Refs. 1 and 2). PPAR␥ is a master transcriptional regulator of lipid and glucose metabolism (reviewed in Refs. 1-3). Inhibition of PPAR␥ function by inflammatory cytokines may contribute to the loss of insulin sensitivity in obese subjects and loss of fat storage in cancer patients under cachexia. Although TNF-␣ is known to inhibit the ligand-dependent transcriptional activity of PPAR␥, the precise mechanism remains to be fully understood (4 -8). In this study, we addressed this issue by analyzing the molecular mechanism of TNF-␣ action on PPAR␥.The transcriptional activity of PPAR␥ is controlled by DNA binding activity and nuclear receptor cofactors that include corepressors and coactivators. PPARs form heterodimers with the retinoid X receptor (RXR), which is activated by 9-cis retinoic acid (9). It is generally believed that the heterodimer is associated with the nuclear receptor corepressor complex in the absence of PPAR␥ ligand. Upon activation by a ligand, the corepressor complex is replaced by coactivators leading to transcriptional initiation of target genes. The corepressor for PPAR␥ is a protein complex containing HDAC3 (histone deacetylase 3) and SMRT (silencing mediator for retinoic and thyroid hormone receptors) or NCoR (nuclear corepressor). RIP140 (receptor-interacting protein) may also be a component in the corepressor complex (10 -13). The coactivators of PPAR␥ include well established cofactors such as p300/ CBP, p160, and PGC-1 (PPAR␥ coactivator-1) (reviewed in Ref. 14), as well as the relatively new coactivators TRAP220 (thyroid hormone receptor-associated protein 220 or PBP, PPAR␥-binding protein) (15, 16), ARA70 (androgen receptor-associated protein) (17), and PRIP (PPAR␥-interacting protein, ASC-2/RAP250/TRBP/NRC) (18 -21). The coactivator p160 has three isoforms: SRC-1 (steroid receptor coactivator 1, ...

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

The Rel/NF-kappaB 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 IkappaBalpha (IkappaBalphaM). In this report, we show that the inhibition of constitutive NF-kappaB activity, either by ectopic expression of IkappaBalphaM or by treating the cells with a proteasome inhibitor PS-341 which blocks intracellular degradation of IkappaBalpha proteins, downregulates the expression of bcl-xl. We identified two putative NF-kappaB binding sites (kappaB/A and B) in the bcl-xl promoter and found that these two sites interact with different NF-kappaB proteins. p65/p50 heterodimer interacts with kappaB/A site whereas p50/p50 homodimer interacts with kappaB/B. The bcl-xl promoter reporter gene assays reveal that NF-kappaB dependent transcriptional activation is mainly mediated by kappaB/A site, indicating that bcl-xl is one of the downstream target genes regulated by RelA/p50. Both IkappaBalphaM and PS-341 completely abolish NF-kappaB DNA binding activity; however, PS-341, but not ectopic expression of IkappaBalphaM, sensitized cells to apoptosis induced by Taxol. This is due to the Taxol-mediated reactivation of RelA through phosphorylation and degradation of IkappaBbeta and the re-expression of NF-kappaB 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-kappaB/IkappaB complexes in regulating anti-apoptotic genes in response to apoptotic stimuli, and they raise the possibility that NF-kappaB : IkappaBalpha and NF-kappaB : IkappaBbeta complexes are regulated by different upstream activators, and that NF-kappaB plays a key role in pancreatic tumorigenesis.

Both pro-and antiapoptotic activities of NF-B transcription factor have been observed; however, less is known about the mechanism by which NF-B induces apoptosis. To elucidate how NF-B regulates proapoptotic signaling, we performed functional analyses using wild-type, ikk1؊/؊ , ikk2 ؊/؊ , rela ؊/؊ murine fibroblasts, MDAPanc-28/Puro, MDAPanc-28/IB␣M, and HCT116/p53 ؉/؉ and HCT116/p53 ؊/؊ cells with investigational anticancer agent doxycycline as a superoxide inducer for generating apoptotic stimulus. In this report, we show that doxycycline increased superoxide generation and subsequently activated NF-B, which in turn up-regulated p53 expression and increased the stability and DNA binding activity of p53. Consequently, NF-Bdependent p53 activity induced the expression of p53-regulated genes PUMA and p21 waf1 as well as apoptosis. Importantly, lack of RelA, IKK, and p53 as well as expression of a dominant negative IB␣ (IB␣M) inhibited NF-B-dependent p53 activation and apoptosis. The doxycycline-induced NF-B activation was not inhibited in HCT116/p53 ؊/؊ cells. Our results demonstrate that NF-B plays an essential role in activation of wild-type p53 tumor suppressor to initiate proapoptotic signaling in response to overgeneration of superoxide. Thus, these findings reveal a mechanism of NF-B-regulated proapoptotic signaling.

We previously reported that NF-B is constitutively activated in most human pancreatic cancer tissues and cell lines but not in normal pancreatic tissues and immortalized pancreatic ductal epithelial cells. IB␣M-mediated inhibition of constitutive NF-B activity in human pancreatic cancer cells suppressed tumorigenesis and liver metastasis in an orthotopic nude mouse model, suggesting that constitutive NF-B activation plays an important role in pancreatic tumor progression and metastasis. However, the underlying mechanism by which NF-B is activated in pancreatic cancer remains to be elucidated. In this study, we found that an autocrine mechanism accounts for the constitutive activation of NF-B in metastatic human pancreatic cancer cell lines. Further investigation showed that interleukin-1␣ was the primary cytokine secreted by these cells that activates NF-B. Neutralization of interleukin-1␣ activity suppressed the constitutive activation of NF-B and the expression of its downstream target gene, urokinasetype plasminogen activator, in metastatic pancreatic cancer cell lines. Our results demonstrate that regulation of interleukin-1␣ expression is primarily dependent on AP-1 activity, which is in part induced by signaling pathways that are epidermal growth factor receptor-dependent and -independent. In conclusion, our findings suggest a possible mechanism for the constitutive activation of NF-B in metastatic human pancreatic cancer cells and a possible missing mechanistic link between inflammation and cancer.