4790

NFKB1

nuclear factor of kappa light polypeptide gene enhancer in B-cells 1;NFKB1;nuclear factor of kappa light polypeptide gene enhancer in B-cells 1

DNA binding factor KBF1|DNA-binding factor KBF1|NF-kappabeta|nuclear factor NF-kappa-B p105 subunit|nuclear factor NF-kappa-B p50 subunit|nuclear factor kappa-B DNA binding subunit

4q24

protein-coding

PURPOSE: Interleukin (IL)-8 is an important mediator of angiogenesis, tumorigenicity, and metastasis in transitional cell carcinoma (TCC) of the bladder. Nuclear factor kappaB (NF-kappaB)/relA regulates IL-8 expression in several neoplasms. The purpose of this study was to determine whether the organ microenvironment (hypoxia, acidosis) regulates the expression of IL-8 in TCC via NF-kappaB, and whether inhibition of NF-kappaB function by mutant IkappaB-alpha prevents induction of IL-8 expression. EXPERIMENTAL DESIGN: IL-8 mRNA expression and protein production by human TCC cell lines (UM-UC-14, HTB-9, RT-4, KU-7 and 253J B-V) were measured by Northern blot analysis and ELISA under acidic (pH 7.35-6.0) and hypoxic (1.0% O(2)) conditions. The involvement of NF-kappaB and activator protein 1 in the regulation of IL-8 production was evaluated by electrophoretic mobility shift assay. Furthermore, the tumorigenicity and metastatic potential of UM-UC-14 cells were determined after transfection with mutant IkappaB-alpha. RESULTS: We found that acidic and hypoxic conditions increased IL-8 mRNA expression and protein production by several, but not all, TCC cell lines evaluated. NF-kappaB, but not activator protein 1, was inducibly activated in UM-UC-14 under both acidic and hypoxic conditions, but not in UM-UC-14 mutant IkappaB-alpha transfectants. tumor growth and lymph node metastasis were inhibited in UM-UC-14 mutant IkappaB-alpha transfectants compared with UM-UC-14 controls. This effect was associated with the inhibition of IL-8 production, cellular proliferation, and angiogenesis. CONCLUSIONS: These results suggest that TCCs of the bladder have heterogenic responses to physicochemical changes in the microenvironment and identify NF-kappaB as a potential molecular target for therapy.

Genomic characterization is beginning to define a molecular taxonomy for breast cancer; however, the molecular basis of invasion and metastasis remains poorly understood. We report a pivotal role for the fibroblast growth factor-inducible 14 (Fn14) receptor in this process. We examined whether Fn14 and its ligand tumor necrosis factor-like weak inducer of apoptosis (TWEAK) were expressed in breast tumors and whether deregulation of Fn14 levels affected malignant behavior of breast cancer cell lines. Analysis of TWEAK and Fn14 in publicly available gene expression data indicated that high Fn14 expression levels significantly correlated with several poor prognostic indicators (P < 0.05). Fn14 expression was highest in the HER2-positive/estrogen receptor-negative (HER2(+)/ER(-)) intrinsic subtype (P = 0.0008). An association between Fn14 and HER2 expression in breast tumors was confirmed by immunohistochemistry. Fn14 levels were elevated in invasive, ER(-) breast cancer cell lines. Overexpression of Fn14 in weakly invasive MCF7 and T47D cells resulted in a marked induction of invasion and activation of nuclear factor-kappaB (NF-kappaB) signaling. Ectopic expression of Fn14tCT, a Fn14 deletion mutant that cannot activate NF-kappaB signaling, was not able to induce invasion. Moreover, ectopic expression of Fn14tCT in highly invasive MDA-MB-231 cells reduced their invasive capability. RNA interference-mediated inhibition of Fn14 expression in both MDA-MB-231 and MDA-MB-436 cells reduced invasion. expression profiling of the Fn14-depleted cells revealed deregulation of NF-kappaB activity. Our findings support a role for Fn14-mediated NF-kappaB pathway activation in breast tumor invasion and metastasis.

Here, we show that NF-kappaB-HIF-1 interaction contributed to breast cancer metastatic capacity by means of an incomplete epithelial/mesenchymal transition and influencing migration, as shown in 1833 (human) and 4T1 (mouse) metastatic cells after different stimuli. The 1833 and the transforming growth factor-beta1-exposed 4T1 cells showed both epithelial (E-cadherins) and mesenchymal (N-cadherins and vimentin) markers, and common mechanisms contributed to the retention of certain epithelial characteristics and the control of migration. The complex NF-kappaB-HIF-1 reciprocal regulation and the enhanced c-Jun expression played a functional role in exacerbating the invasiveness of 1833 cells after p50/p65 transfection and of 4T1 cells exposed to transforming growth factor-beta1. Twist expression seemed to exert a permissive role also regulating epithelial/mesenchymal transition markers. After c-Src wild-type (Srcwt) transfection, c-Src-signal transducer overexpression in 1833 cells increased HIF-1 transactivating activity and invasiveness, and changed E-cadherin/N-cadherin ratio versus mesenchymal phenotype. The transcription factor pattern and the motile phenotype of metastatic 1833 cells were influenced by p65-lysine acetylation and HDAC-dependent epigenetic mechanisms, which positively regulated basal NF-kappaB and HIF-1 activities. However, HDAC3 acted as a corepressor of NF-kappaB activity in parental MDA-MB231 cells, thus explaining many differences from the derived 1833 clone, including reduced HIF-1alpha and c-Jun expression. Invasiveness was differently affected by HDAC knockdown in 1833 and MDA-MB231 cells. We suggest that acetylation/deacetylation are critical in establishing the bone-metastatic gene signature of 1833 cells by regulating the activity of NF-kappaB and HIF-1, and further clarify the epigenetic control of transcription factor network in the motile phenotype of 1833 cells.

NF-kappaB activation induced by genotoxic treatment in cancer cells has been associated with therapeutic resistance in multiple human malignancies. Therapeutic resistance also correlates with high metastatic potential in human cancers, including breast cancer. Whether genotoxic treatment-activated NF-kappaB also contributes to cancer metastasis following radiation and chemotherapy is unclear. Here, we show that chemotherapeutic drug-induced NF-kappaB activation promotes breast cancer cell migration and invasion. The increased metastatic potential is dependent on IL-6 induction mediated by genotoxic NF-kappaB activation. Moreover, genotoxic treatment also up-regulates oncogenic microRNA-21 (miR-21) expression through eliciting NF-kappaB recruitment to the miR-21 promoter region, where it cooperates with signal transducer and activator of transcription 3 (STAT3) to activate miR-21 transcription. DNA damage-induced histone H3 phosphorylation via activated MSK1 creates an open chromatin structure for NF-kappaB/STAT3-driven transactivation of miR-21. NF-kappaB-dependent IL-6 up-regulation is responsible for STAT3 activation and recruitment to the miR-21 promoter upon genotoxic stress. Induction of miR-21 may enable cancer cells to elude DNA damage-induced apoptosis and enhance the metastatic potential of breast cancer cells through repressing expression of PTEN and PDCD4. Our data support a critical role of DNA damage-induced NF-kappaB activation in promoting cancer metastasis following genotoxic treatment, and NF-kappaB-dependent miR-21 induction may contribute to both therapeutic resistance and metastasis in breast cancer.

The transcription factor NFkappaB regulates the expression of several tumor-related molecules associated with tumor progression and metastasis. However, the precise mechanisms by which its activation mediates these processes in diverse tumors are unknown. In this study we determined the expression of NFkappaB in various colorectal carcinoma cell lines, in a series of 90 non-metastatic and metastatic colorectal tumors and in an in vitro 3D-spheroid model of HT-29 cells simulating morphological hallmark of these adenocarcinomas, namely neoplastic glandular nests around a necrotic center. We show that the inactive cytoplasmic NFkappaB form is evidently up-regulated in the tumor epithelium, especially in the metastatic cases, as compared to normal tissue. We found that in situ nuclear NFkappaB staining is characteristic for cells that are still viable but dissociated from the surrounding cohesive tumor tissue and destined to die. Evidence for a possible association between NFkappaB expression and loss of cell adhesion mediated by E-cadherin function has been provided in vivo and in vitro using the HT-29 3D-spheroid model. In both cases, we found a strong correlation between activation of NFkappaB and loss of E-cadherin expression. Considering the fact that cancer cell necrosis plays a crucial role in metastasis, NFkappaB activation mediated by loss of E-cadherin may represent an essential, even initial event in this process. Furthermore, we present in vitro data implicating LPS, the endotoxin of gram-negative bacteria, in the triggering of NFkappaB up-regulation. Thus, release of bacterial endotoxin may essentially contribute to the progression of colon cancer in vivo.

PURPOSE: NF-kappaB is an antiapoptotic transcription factor that has been shown to be a mediator of treatment resistance. Bcl-3 is a regulator of NF-kappaB that may play a role in oncogenesis. The goal of this study was to correlate the activation status of NF-kappaB and Bcl-3 with clinical outcome in a group of patients with metastatic colorectal cancer (CRC). METHODS: A retrospective study of 23 patients who underwent surgical resection of CRC at the University of North Carolina (UNC). Activation of NF-kappaB was defined by nuclear expression of select components of NF-kappaB (p50, p52, p65) and Bcl-3. Tissue microarrays were created from cores of normal mucosa, primary tumor, lymph node metastases and liver metastases in triplicate from disparate areas of the blocks, and an intensity score was generated by multiplying intensity (0-3+) by percent of positive tumor cells. Generalized estimating equations were used to note differences in intensity scores among normal mucosa and nonnormal tissues. Cox regression models were fit to see if scores were significantly associated with overall survival. RESULTS: p65 NE was significantly higher in primary tumor and liver metastases than normal mucosa (both p < 0.01). p50 nuclear expression was significantly higher for all tumor sites than for normal mucosa (primary tumor and lymph node metastases p < 0.0001, liver metastases p < 0.01). Bcl-3 nuclear expression did not differ significantly between normal mucosa and tumor; however, nuclear expression in primary tumor for each of these components was strongly associated with survival: the increase in hazard for each 50-point increase in nuclear expression was 91% for Bcl-3, 66% for p65, and 52% for p50 (all p < 0.05). CONCLUSIONS: Activation of canonical NF-kappaB subunits p50 and p65 as measured by nuclear expression is strongly associated with survival suggesting NF-kappaB as a prognostic factor in this disease. Primary tumor nuclear expression appears to be as good as, or better than, metastatic sites at predicting prognosis. Bcl-3 nuclear expression is also negatively associated with survival and deserves further study in CRC.

BACKGROUND: The transcription factor nuclear factor-kappaB (NF-kappaB) has been implicated in gastric cancer metastasis, but the underlying molecular mechanisms remain unclear. We investigated the role of the interaction between NF-kappaB and signal transducers and activators of transcription 3 (STAT3) in controlling metastatic potential of gastric cancer cells. METHODS: Immunohistochemistry for NF-kappaB p65 (RelA), phospho-Tyr705-STAT3 (pSTAT3), or matrix metalloproteinase 9 (MMP9) was performed on tissue array slides containing 255 gastric carcinoma specimens. NF-kappaB inhibition in SNU-638 and MKN1 gastric cancer cell lines were performed by transduction with a retroviral vector containing NF-kappaB repressor mutant of IkappaBalpha, and STAT3 was silenced by RNA interference. We also did luciferase reporter assay, double immunofluorescence staining and immunoblotting. Cell migration and invasion were determined by wound-healing assay and invasion assay, respectively. RESULTS: NF-kappaB and STAT3 were constitutively activated and were positively correlated (P=0.038) in gastric cancer tissue specimens. In cell culture experiments, NF-kappaB inhibition reduced STAT3 expression and activation, whereas STAT3 silencing did not affect NF-kappaB activation. Moreover, both NF-kappaB inhibition and STAT3 silencing decreased gastric cancer cell migration and invasion in a synergistic manner. In addition, both NF-kappaB activation and STAT3 activation were positively correlated with MMP9 in gastric cancer tissues (P=0.001 and P=0.022, respectively), decreased E-cadherin expression and increased Snail and MMP9 expressions in cultured cells. CONCLUSION: NF-kappaB and STAT3 are positively associated and synergistically contribute to the metastatic potential of gastric cancer cells. Thus, dual use of NF-kappaB and STAT3 inhibitors may enhance the efficacy of the anti-metastatic treatment of gastric cancer.

metastatic ovarian granulosa cell tumors (GCT) exhibit loss of betaglycan. Here we test the hypothesis that betaglycan blocks GCT metastasis by suppressing NFkappaB/TGFbeta2-induced matrix metalloprotinease-2 (MMP2). Human GCT and a human GCT cell model demonstrated prominent MMP2 expression, which was dependent on NFkappaB activity and stimulated by TGFbeta2 in an NFkappaB-dependent manner. Betaglycan suppressed both basal and TGFbeta2-induced MMP2 expression and countered metastatic behaviors of GCT cells in non-adherent spheroid culture and in vivo xenograft models of metastasis. These data suggest that NFkappaB/TGFbeta2 promotes, and betaglycan impedes, the early stages of GCT metastasis, when tumor cells first invade the peritoneum.

BACKGROUND: An increasing incidence of cholangiocarcinoma (CCA) and CCA mortality rates has been observed around the world. Patients with intrahepatic biliary stones have a 10% risk of developing CCA, and up to 70% of patients with histologically confirmed CCA have hepatolithiasis. Few previous studies have addressed the associations between the expression of nuclear factor (NF)-kappaB and epidermal growth factor receptor (EGFR) and clinicopathological prognosis in patients with hepatolithiasis-associated intrahepatic CCA. AIMS: This study was designed to investigate the clinicopathological and prognostic significance of NF-kappaB and EGFR expression in hepatolithiasis-associated intrahepatic CCA and hepatolithiasis. METHODS: A total of 90 liver specimens were immunohistochemically stained for NF-kappaB and EGFR expression, and the characteristics of 90 individual patients were retrospectively reviewed. RESULTS: Differences in the positive rates of NF-kappaB and EGFR expression between the hepatolithiasis-associated intrahepatic CCA group, intrahepatic lithiasis group, and control group were found to be statistically significant. EGFR expression was closely associated with the degree of differentiation and depth of invasion (p < 0.05). The 1-, 3-, and 5-year overall survival rates were respectively 42.8, 21.0, and 10.3% in intrahepatic CCA groups. The survival rate of the EGFR-negative group was higher than in the EGFR-positive group (p < 0.01). Lymph node metastasis (HR 1.24, 95%

BACKGROUND: During the process of metastasis, cells are subjected to various apoptotic stimuli. Aberrant expression of apoptotic regulators often contribute to cell metastasis. Heat shock protein 27(HSP27) is confirmed as an apoptosis regulator, but its antiapoptotic mechanism in metastatic hepatocellular carcinoma (HCC) cells remains unclear. METHODS: Levels of HSP27 protein and its phosphorylation in Hep3B, MHCC97L to MHCC97H cells with different metastatic potentials were determined by western blot analysis. MHCC97H cells were transfected with specific small interference RNA (siRNA) against HSP27. The in vitro migration and invasion potentials of cells were evaluated by Transwell assay. The apoptosis ratio of MHCC97H cells was analyzed by TUNEL staining and Flow Cytometry. Alteration of signal transduction pathway after HSP27 knockdown in MHCC97H cells was evaluated through a Human Q Series Signal Transduction in cancer Gene Array analysis. Nuclear NF-kappaB contentration and endogenous IKK activity were demonstrated by ELISA assay. The association of IKKalpha, IKKbeta, IkappaBalpha with HSP27 and the association between IKKbeta and IKKalpha in MHCC97H cells were determined by co-immunoprecipitation assay followed by western blot analysis. RESULTS: HSP27 protein and its phosphorylation increased in parallel with enhanced metastatic potentials of HCC cells. siRNA-mediated HSP27 knockdown in MHCC97H significantly suppressed cells migration and invasion in vitro and induced cell apoptosis; the prominently altered signal transduction pathway was NF-kappaB pathway after HSP27 knockdown in MHCC97H cells. Furthermore, inhibition of HSP27 expression led to a significant decrease of nuclear NF-kappaB contentration and endogenous IKK activity. In addition, HSP27 was associated with IKKalpha, IKKbeta, IkappaBalpha in three HCC cells above. ELISA assay and western blot analysis also showed a decrease of the association between IKKbeta and IKKalpha, the association between phosphor-HSP27 and IKK complex, and an increase of total IkappaBalpha but reducing tendency of phosphor-IkappaBalpha when HSP27 expression was efficiently knocked down in MHCC97H cells. CONCLUSION: Altogether, these findings revealed a possible effect of HSP27 on apoptosis in metastatic HCC cells, in which HSP27 may regulate NF-kB pathway activation.

Hepatitis B virus X protein (HBx) promotes hepatocellular carcinoma (HCC) invasion and metastasis by a poorly understood mechanism. This study investigated the role of NF-kappa B in HBx-mediated upregulation of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). In a stably expressing HBx cell line, NF-kappa B level was examined by laser scanning confocal microscopy before and after treatment with pyrrolidine dithiocarbamate (PDTC; an NF-kappa B inhibitor). VEGF, MMP2, MMP9, and MMP14 mRNA and protein levels were quantitated by real-time PCR and Western blotting, respectively. HBx stimulated NF-kappa B signaling and increased VEGF, MMP2, MMP9, and MMP14 mRNA and protein levels. PDTC treatment blocked HBx-mediated stimulation of NF-kappa B signaling and decreased VEGF, MMP9, and MMP14 (but not MMP2) mRNA and protein levels. In vivo studies, PDTC reduced angiogenesis in subcutaneous xenograft of nude mice which injected HepG2-HBx cells. This suggests that NF-kappa B is involved in upregulating these genes and in the HBx-mediated invasion and metastasis of HCC.

BACKGROUND AND OBJECTIVES: We previously showed that the calcium-binding protein S100A4 is overexpressed and related to metastasis in hepatocellular carcinoma (HCC). However, whether S100A4 participates in the regulation of metastasis and its mechanisms in HCC is mostly unknown. Given the associations of S100A4, nuclear factor-kB (NF-kB/RelA) and MMP-9 with metastasis in a variety of malignancies, we explored a potential role of S100A4 in HCC metastasis and its mechanism. METHODS: 20 patients with HCC invasion (Lymph node metastasis, microvascular invasion, major portal vein invasion and intrahepatic metastasis) and 20 patients without HCC invasion were included. These tissues were detected for the expression of S100A4, NF-kB/RelA and MMP-9 by immunohistochemistry and quantitative real time polymerase chain reaction (Q-PCR). Correlation between the expressions of S100A4, NF-kB/RelA and MMP-9 with the invasion was analysed. The expressions of S100A4, nuclear factor-kB and MMP-9 was evaluated in HepG2 cells by western blot and immunohistochemistry. HepG2 cells were stably transfected with S100A4-specific small interfering RNA (S100A4 siRNA) to knockdown of S100A4, then transiently transfected with S100A4 cDNA to rescure the S100A4 level and evaluated for effects on invasion and expression analysis for molecules involved in invasion. After the HepG2 cells recurred the S100A4 levels, the HepG2 cells was treated with 5 microM Pyrrolidine Dithiocarbamate (PDTC) (a selective NF kappa B inhibitor) to inhibit the NF-kB activity, or treated with Batimast (BB94: a MMPs inhibitor) to inhibit the MMP-9 activity. The expression analysis for molecules involved in invasion was analyzed. RESULTS: A significant increase of S100A4, NF-kB/RelA and MMP-9 expression in HCC tissues with invasion than that of without invasion. A positive correlation was observed between S100A4, NF-kB/RelA, MMP-9 and invasion, respectively. In addition, S100A4 was positively correlated with NF-kB and MMP-9. S100A4 siRNA mediated knockdown of S100A4 in HepG2 cells resulted in significant reduction in the NF-kB activity and MMP-9 expression, and dramatically decreased its invasion. Moreover, the HepG2 cell metastatic potential was rescued by overexpression of S100A4 completely, at the same time, the NF-kB activity and MMP-9 expression was also increased. Pretreatment with PDTC or BB94 was observed to significantly reduce NF-kB activity and MMP-9 expression and dramatically decreased S100A4 -induced invasion. CONCLUSIONS: Our findings indicate that S100A4 contributes to HCC metastasis by activation of NF-kB dependent MMP-9 expression, suggesting S100A4 as a novel diagnostic biomarker and therapeutic target in HCC.

Nuclear factor kappa B (NFkappaB) is an essential regulator of gene transcription for hundreds of genes, including many critically involved in apoptosis. NFkappaB complexes containing cRel generally activate pro-apoptotic genes, while those with RelA activate anti-apoptotic genes. We have previously shown that NFkappaB binding by RelA is constitutively elevated in human metastatic melanoma cultures relative to normal melanocytes. Here we extended our investigation to immunohistochemical analysis of human tissue biopsies. We found that RelA expression is significantly elevated in melanocytes of human naevi and melanomas relative to normal skin, but expression of its inhibitor IkappaB-alpha is significantly lower in metastatic melanomas than in intradermal naevi. Antibodies specific for the nuclear localization signal of RelA also showed significantly increased staining in metastatic melanoma biopsies. Notably, in melanomas and in naevi, we also found that RelA is phosphorylated at serine 529, and this activated form accumulates in the nuclei of melanomas. This suggests that increased expression and phosphorylation of RelA occurs at the stage of the benign naevus, but IkappaB-alpha is able to sequester RelA in the cytoplasm and regulate RelA transcriptional transactivation. We also found that antibodies against cRel show a progressive increase in staining from naevi to melanoma. However, staining for IkappaB-epsilon, which primarily inhibits the nuclear localization of cRel was also progressively increased, and cRel expression was predominantly cytoplasmic in melanomas. These results confirm that the altered expression of RelA found in metastatic melanoma cells in tissue culture is relevant to human tumors and offer new insights into the deregulation of NFkappaB signaling.

Autotaxin, a lysophospholipase D producing lysophosphatidic acid, augments invasive and metastatic potential of tumor cells. Current investigations have focused on understanding the molecular mechanisms by which autotaxin regulates the expression of a major mediator of tumor invasion and metastasis, urokinase-type plasminogen activator (uPA) in human A2058 melanoma cells. Autotaxin induced uPA expression in a dose-dependent manner that was inhibited by pharmacological inhibitors for Gi (pertussis toxin), phosphoinositide 3-kinase (PI3K, LY294002), Akt inhibitor (AktI), proteosome activity and IkappaB phosphorylation (pyrrolidine dithiocarbamate), and by a dominant negative mutant (DN) of Akt. Autotaxin phosphorylated Akt and induced the translocation of nuclear [corrected] factor-kappaB (NF-kappaB) to the nucleus that were inhibited by AktI or by overexpressing DN-Akt. Consistently, green fluorescence protein-tagged p65 of NF-kappaB accumulated in the nucleus by autotaxin that was abrogated when the cells were transfected with DN-Akt. Moreover, autotaxin increased the DNA binding ability of NF-kappaB and promoter activity of uPA. Collectively, these data strongly suggest autotaxin induces uPA expression via the Gi-PI3K-Akt-NF-kappaB signaling pathway that might be critical for autotaxin-induced tumor cell invasion and metastasis.

MicroRNAs (miRNAs) are short non-coding RNAs that post-transcriptionally regulate gene expression. Hsa-miR-9 has been shown to have opposite functions in different tumour types; however, the underlying mechanism is unclear. Here we show that hsa-miR-9 is down-regulated in metastatic melanomas compared to primary melanomas. Overexpression of miR-9 in melanoma cells resulted in significantly decreased cell proliferation and migratory capacity with decreased F-actin polymerization and down-regulation of multiple GTPases involved in cytoskeleton remodelling. miR-9 overexpression induced significant down-regulation of Snail1 with a concomitant increase in E-cadherin expression. In contrast, knockdown of miR-9 increased Snail1 expression as well as melanoma cell proliferation and migration capacity. Mechanistically, miR-9 expression down-regulated NF-kappaB1 in melanoma and the effect was abolished by mutations in the putative miR-9 binding sites within the 3'-untranslated region (UTR) of NF-kappaB1. Anti-miR-9 miRNA inhibitor also increased the expression of NF-kappaB1. The effects of miR-9 on Snail1 expression and melanoma cell proliferation and migration were rescued by overexpression of NF-kappaB1 in these cells. Furthermore, miR-9 overexpression resulted in significantly decreased melanoma growth and metastasis in vivo. In summary, miR-9 inhibits melanoma proliferation and metastasis through down-regulation of the NF-kappaB1-Snail1 pathway. This study finds a new mechanism that miR-9 utilizes to decrease E-cadherin expression and inhibit melanoma progression. The results suggest that function of microRNAs is context and tumour type-specific.

The increased motility and invasiveness of tumor cells are reminiscent of epithelial-mesenchymal transition (EMT), which occurs during embryonic development, wound healing, and metastasis. In this study, we found that Snail is stabilized by the inflammatory cytokine TNFalpha through the activation of the NF-kappaB pathway. We demonstrated that NF-kappaB is required for the induction of COP9 signalosome 2 (CSN2), which, in turn, blocks the ubiquitination and degradation of Snail. Furthermore, we showed that the expression of Snail correlated with the activation of NF-kappaB in cancer cell lines and metastatic tumor samples. Knockdown of Snail expression inhibited cell migration and invasion induced by inflammatory cytokines and suppressed inflammation-mediated breast cancer metastasis. Our study provides a plausible mechanism for inflammation-induced metastasis.

Nuclear factor (NF)-kappaB is frequently over-expressed in non-small cell lung cancer (NSCLC), but the exact role of this observation remains unclear. In this regard, activation of the transcription factor may govern distinct steps of NSCLC progression, such as carcinogenesis, angiogenesis, and metastasis. In these studies we attempted to dissect the effects of two proteins of the NF-kappaB pathway (p65/RelA and IkappaBetaalpha) on experimental metastasis of murine NSCLC, using a novel approach of bioluminescent detection of NF-kappaB activation in tumor cells. Stable integration of a NF-kappaBeta reporter confirmed high basal activation of the transcription factor in mouse NSCLC cells in vitro and during experimental metastasis to the lungs, like human NSCLC. In the mouse model of NSCLC metastasis, NF-kappaBeta-dependent luciferase expression served as a reliable indicator of tumor cell delivery to the lungs, establishment of metastatic tumors, and lung tumor burden. In vitro transient p65/RelA and IkappaBetaalpha gene transfer to mouse NSCLC cells resulted, respectively, in significant NF-kappaB activation and inhibition, without affecting cell growth. However, p65/RelA overexpression in NSCLC cells drastically reduced in vivo metastasis to the lungs, while overexpression of IkappaBetaalpha had no effect. In conclusion, using bioluminescent detection of NF-kappaB activation in mouse lug adenocarcinoma cells, we found a negative impact of p65/RelA on NSCLC metastasis.

The epithelial-to-mesenchymal transition (EMT) is a de-differentiation process that has been implicated in metastasis and the generation of cancer initiating cells (CICs) in solid tumors. To examine EMT in non-small cell lung cancer (NSCLC), we utilized a three dimensional (3D) cell culture system in which cells were co-stimulated with tumor necrosis factor alpha (TNF) and transforming growth factor beta (TGFbeta). NSCLC spheroid cultures display elevated expression of EMT master-switch transcription factors, TWIST1, SNAI1/Snail1, SNAI2/Slug and ZEB2/Sip1, and are highly invasive. Mesenchymal NSCLC cultures show CIC characteristics, displaying elevated expression of transcription factors KLF4, SOX2, POU5F1/Oct4, MYCN, and KIT. As a result, these putative CIC display a cancer "stem-like" phenotype by forming lung metastases under limiting cell dilution. The pleiotropic transcription factor, NF-kappaB, has been implicated in EMT and metastasis. Thus, we set out to develop a NSCLC model to further characterize the role of NF-kappaB activation in the development of CICs. Here, we demonstrate that induction of EMT in 3D cultures results in constitutive NF-kappaB activity. Furthermore, inhibition of NF-kappaB resulted in the loss of TWIST1, SNAI2, and ZEB2 induction, and a failure of cells to invade and metastasize. Our work indicates that NF-kappaB is required for NSCLC metastasis, in part, by transcriptionally upregulating master-switch transcription factors required for EMT.

Areca (betel)-chewing is tightly associated with the high prevalence of oral squamous cell carcinoma (OSCC) in Asians. NFKB1 encodes a 105kDa protein that can be processed to produce p50 subunit of nuclear factor-kappaB protein complex. A insertion (ins)/deletion (del) polymorphism (-94ins/delATTG) in NFKB1 promoter, which may drive the ins allele two-fold increase in NFKB1 transcription relative to del allele, was recently found. This study identified that the odds ratio in OSCC carrying ins allelotype were 1.78 relative to controls (56.7 vs 41.8%) in subjects more than 50 years old. L allelotype of Heme oxygenase-1 (HO-1), accounting for a long (GT)(n) repeat in HO-1 promoter, is associated with the risks of areca-related OSCC. Subjects carried both NFKB1 ins and HO-1 L allelotypes had significant risks for various subsets of OSCC. OSCC with lymph node metastasis or advanced stage had significantly higher frequency of NFKB1 ins and HO-1 L allelotypes. This study suggested that the functional NFKB1promoter polymorphism could be valuable for assessment of cancer risk.

The poor prognosis of human ovarian cancer is partly due to its metastasis and recurrence. It has been demonstrated that tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK)-fibroblast growth factor inducible-14 (Fn14) signaling system may be a potential regulator of human tumorigenesis. The objective of this study was to understand the effect of TWEAK on ovarian cancer metastasis. We recently showed that activation of Fn14 signaling by TWEAK promoted cell migration and invasion in human HO-8910PM cells. Treating HO-8910PM cells with TWEAK resulted in the activation of nuclear factor-kappa B (NF-kappaB) and subsequently the translocation of NF-kappaB from cytoplasm to nucleus. In addition, TWEAK promoted vascular endothelial growth factor (VEGF) protein expression, and this effect was dependent upon NF-kappaB transcriptional activity. Blocking the NF-kappaB pathway with PDTC suppressed TWEAK-induced up-regulation of VEGF protein expression and cell metastasis. Our results suggest that TWEAK-Fn14 functions, in part, through the NF-kappaB signaling pathway to up-regulate VEGF expression to foster ovarian cancer cell metastasis. Targeted therapy against TWEAK-Fn14 signaling system as an adjuvant to surgery may improve clinical management of invasive ovarian cancer cells and advance the outcome of this devastating cancer.

metastatic ovarian granulosa cell tumors (GCT) exhibit loss of betaglycan. Here we test the hypothesis that betaglycan blocks GCT metastasis by suppressing NFkappaB/TGFbeta2-induced matrix metalloprotinease-2 (MMP2). Human GCT and a human GCT cell model demonstrated prominent MMP2 expression, which was dependent on NFkappaB activity and stimulated by TGFbeta2 in an NFkappaB-dependent manner. Betaglycan suppressed both basal and TGFbeta2-induced MMP2 expression and countered metastatic behaviors of GCT cells in non-adherent spheroid culture and in vivo xenograft models of metastasis. These data suggest that NFkappaB/TGFbeta2 promotes, and betaglycan impedes, the early stages of GCT metastasis, when tumor cells first invade the peritoneum.

We previously reported that NF-kappaB is constitutively activated in most human pancreatic cancer tissues and cell lines but not in normal pancreatic tissues and immortalized pancreatic ductal epithelial cells. IkappaBalphaM-mediated inhibition of constitutive NF-kappaB activity in human pancreatic cancer cells suppressed tumorigenesis and liver metastasis in an orthotopic nude mouse model, suggesting that constitutive NF-kappaB activation plays an important role in pancreatic tumor progression and metastasis. However, the underlying mechanism by which NF-kappaB 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-kappaB in metastatic human pancreatic cancer cell lines. Further investigation showed that interleukin-1alpha was the primary cytokine secreted by these cells that activates NF-kappaB. Neutralization of interleukin-1alpha activity suppressed the constitutive activation of NF-kappaB and the expression of its downstream target gene, urokinase-type plasminogen activator, in metastatic pancreatic cancer cell lines. Our results demonstrate that regulation of interleukin-1alpha 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-kappaB in metastatic human pancreatic cancer cells and a possible missing mechanistic link between inflammation and cancer.

Emodin (1, 3, 8-trihydroxy-6-methylanthraquinone) is an active constituent isolated from the root of Rheum palmatum L and is the main effective component of some Chinese herbs and plants. Pharmacological studies have demonstrated that emodin exhibits anti-cancer effects on several human cancers. However, the molecular mechanisms of emodin-mediated tumor regression have not been fully defined. This study was performed to investigate the antiproliferative and antimetastatic effects of emodin on pancreatic cancer in vitro and in vivo. Our results showed that emodin induced a higher percentage of growth inhibition and apoptosis in the pancreatic cancer cell line SW1990 compared to that of control, and emodin suppressed the migration and invasion of SW1990 cells in a dose-dependent manner. To investigate the possible mechanisms involved in these events, we performed electrophoretic mobility shift assay (EMSA) and Western blot analysis, and found that emodin significantly down-regulated NF-kappaB DNA-binding activity, survivin and MMP-9 in SW1990 cells. Moreover, the expression of cleaved caspase-3 was up-regulated in SW1990 cells after treatment with emodin. In addition, a metastatic model simulating human pancreatic cancer was established by orthotopic implantation of histologically intact human tumor tissue into the pancreatic wall of nude mice. Oral administration of emodin significantly decreased tumor weight and metastasis compared to control. Furthermore, the expression of NF-kappaB, survivin and MMP-9 were also suppressed in tumor tissues after treatment with emodin. Collectively, our results indicated that emodin exerts antiproliferative and antimetastatic activity on pancreatic cancer both in vitro and in vivo, which may be related to down-regulation of NF-kappaB and its regulated molecules such as survivin and MMP-9 proteins. Consequently, these results provide important insights into emodin as an anti-invasive agent for the therapy of human pancreatic cancer.

INTRODUCTION: Nuclear factor-kappaB (NF-kappaB) is a transcription factor that transactivates genes involved in the regulation of cell growth, apoptosis, angiogenesis, and metastasis. Our aim was to assess NF-kappaB expression in lymph node (LN) metastases of prostate cancer. METHODS: Immunohistochemical staining was performed using the p65 anti-NF-kappaB antibody. Seventy-seven paraffin-embedded LN specimens obtained from 54 prostate cancer patients were analyzed. Of the 54 patients, 32 had positive LN metastases, while 22 showed no evidence of metastasis and were considered as controls. The overall percentage of NF-kappaB-nuclear localization was assessed, as well as the intensity of staining. RESULTS: Nuclear localization of NF-kappaB was significantly greater in the metastatic LN group compared to controls. In patients with positive-LN metastases, 84.4% showed >10% nuclear staining in tumor cells. Moreover, 64.4% of the malignant LN specimens had >10% nuclear staining in lymphocytes compared to 0% in controls. Intensity of cytoplasmic and nuclear staining was higher in the metastatic LN group than in controls (P < 0.01). CONCLUSIONS: Nuclear localization/activation of NF-kappaB is up-regulated in prostate cancer LN metastasis. Such up-regulation of NF-kappaB activity is observed in the tumor cells as well as in the surrounding lymphocytes.

CpG-oligonucleotides (CpG-ODNs), mimicking bacterial DNA, have recently been shown to stimulate prostate cancer invasion in vitro via Toll-like receptor 9 (TLR9). Since cyclooxygenase 2 (COX-2), frequently overexpressed in multiple tumor types including prostate cancer, is a causal factor for tumor development, invasion and metastasis, an interesting question is raised whether TLR9 regulates COX-2 expression in prostate cancer cells. To address this question, herein we examined COX-2 expression in PC-3 cells stimulated with different doses and time courses of CpG-ODNs. The regulatory role of NF-kappaB in TLR9-mediated COX-2 expression was also investigated. CpG-ODN was found to up-regulate the expression of COX-2 in PC-3 cells in a dose- and time-dependent manner, but have little impact on COX-1 expression. Moreover, CpG-ODN also promoted nuclear translocation and activation of NF-kappaB, which appeared to be required for COX-2 induction by CpG-ODN. Overall, TLR9 up-regulates COX-2 expression in prostate cancer cells, at least partially through the activation of NF-kappaB, which may be implicated in tumor invasion and metastasis.

The molecular mechanisms involved in prostate cancer (PC) metastasis and bone remodeling are poorly understood. We recently reported that phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) mediates transcriptional regulation and activation of bone morphogenetic protein (BMP)-2 signaling by nuclear factor (NF)-kappaB in bone metastatic prostate cancer cells. In the present study, we demonstrate that NF-kappaB, whether activated by recombinant human tumor necrosis factor (TNF)-alpha or by ectopic expression of the p65 subunit, is involved in extracellular matrix adhesion and invasion of osteotropic PC-3 and C4-2B, but not LNCaP, cells. The enhanced metastatic potential was associated with transcriptional upregulation of osteopontin, osteocalcin, and collagen IA1 in osteotropic PC cells, suggesting their role in osteomimicry of PC cells. Unlike BMP-4, BMP-2 protein enhanced the invasive properties of C4-2B cells, but not in LNCaP cells. Also, this effect was nullified by Noggin. In addition, BMP-2 mediates TNF-alpha-induced invasion of C4-2B cells in a NF-kappaB-dependent fashion. TNF-alpha or conditioned media (CM) of TNF-alpha-stimulated C4-2B cells upregulated BMP-2 and BMP-dependent Smad transcripts and inhibited receptor activator of NF-kappaB ligand transcripts in RAW 264.7 preosteoclast cells, respectively, implying that this factor may contribute to suppression of osteoclastogenesis via direct and paracrine mechanisms. In contrast, CM of TNF-alpha-stimulate or BMP2-stimulated C4-2B cells induced in vitro mineralization of MC3T3-E1 osteoblast cells in a BMP-2-dependent and NF-kappaB-dependent manner, respectively. Taken together, the results suggest that mutual interactions between these factors may be pivotal not only in enhancing the osteomimicry and metastatic potential of PC cells, but also in bone remodeling and in shifting the balance from osteoclastogenesis towards osteoblastogenesis.

Patients with advanced prostate cancer almost invariably develop osseous metastasis. Although many studies indicate that the activation of NF-kappaB signaling appears to be correlated with advanced cancer and promotes tumor metastasis by influencing tumor cell migration and angiogenesis, the influence of altered NF-kappaB signaling in prostate cancer cells within boney metastatic lesions is not clearly understood. While C4-2B and PC3 prostate cancer cells grow well in the bone, LNCaP cells are difficult to grow in murine bone following intraskeletal injection. Our studies show that when compared to LNCaP, NF-kappaB activity is significantly higher in C4-2B and PC3, and that the activation of NF-kappaB signaling in prostate cancer cells resulted in the increased expression of the osteoclast inducing genes PTHrP and RANKL. Further, conditioned medium derived from NF-kappaB activated LNCaP cells induce osteoclast differentiation. In addition, inactivation of NF-kappaB signaling in prostate cancer cells inhibited tumor formation in the bone, both in the osteolytic PC3 and osteoblastic/osteoclastic mixed C4-2B cells; while the activation of NF-kappaB signaling in LNCaP cells promoted tumor establishment and proliferation in the bone. The activation of NF-kappaB in LNCaP cells resulted in the formation of an osteoblastic/osteoclastic mixed tumor with increased osteoclasts surrounding the new formed bone, similar to metastases commonly seen in patients with prostate cancer. These results indicate that osteoclastic reaction is required even in the osteoblastic cancer cells and the activation of NF-kappaB signaling in prostate cancer cells increases osteoclastogenesis by up-regulating osteoclastogenic genes, thereby contributing to bone metastatic formation.

Characteristically, prostate cancer (PCa) cells exhibit marked decrease in intracellular zinc; however, the mechanism responsible is not clearly understood. HOXB13 is involved in PCa progression and is overexpressed in castration-resistant PCa. DNA microarray analysis of LNCaP Pca cells showed that ZnT zinc output transporters were strikingly upregulated among androgen-independent HOXB13 target genes. Furthermore, exogenous HOXB13 caused intracellular zinc concentrations to fall in PCa cells, stimulated NF-kappaB-mediated signaling by reducing inhibitor of NF-kappaB alpha (IkappaBalpha) and enhanced the nuclear translocation of RelA/p65. Human prostate tumors also exhibited strong inverse correlation between the protein expressions of HOXB13 and IkappaBalpha. Consequently, HOXB13 stimulated PCa cell invasion, and this was inhibited by the suppression of ZnT4. In addition, studies in a PC3 orthotopic mouse model of PCa metastasis showed that HOXB13 is a strong metastatic stimulator. Taken together, these results show that HOXB13 promotes PCa invasion and metastasis by decreasing intracellular zinc levels, thus stimulating NF-kappaB signals, and suggest that HOXB13 acts as a modulator of intracellular zinc levels that promotes the malignant characteristics of PCa.