Cylindromatosis and the CYLD gene: new lessons on the molecular principles of epithelial growth control

Analysing cylindromatosis and the associated defects in the CYLD gene is providing novel insights into the molecular principles of epithelial growth control and carcinogenesis in, and beyond, the skin. In this review, we summarize the histopathology and histogenesis of cylindromas, and the available genetic information on patients with these skin appendage tumors. Focusing on recent data concerning the normal functions and signaling interactions of the CYLD gene product, we explain how CYLD interferes with TNF-alpha or TLR-mediated signaling as well as with JNK or NF-kappaB-dependent p65/50 signaling to limit inflammation. In addition, we delineate how CYLD interferes with activation of the proto-oncogene Bcl(3) and with cyclin D1 expression to limit tumorigenesis, and chart how tumor growth-promoting agents or UV light and inflammatory mediators can activate CYLD. We argue that these recent insights into CYLD function and cylindroma pathogenesis may lead to the development of novel molecular strategies for cancer prevention and treatment.     

Mouse mammary tumor virus c-rel transgenic mice develop mammary tumors

Amplification, overexpression, or rearrangement of the c-rel gene, encoding the c-Rel NF-kappaB subunit, has been reported in solid and hematopoietic malignancies. For example, many primary human breast cancer tissue samples express high levels of nuclear c-Rel. While the Rev-T oncogene v-rel causes tumors in birds, the ability of c-Rel to transform in vivo has not been demonstrated. To directly test the role of c-Rel in breast tumorigenesis, mice were generated in which overexpression of mouse c-rel cDNA was driven by the hormone-responsive mouse mammary tumor virus long terminal repeat (MMTV-LTR) promoter, and four founder lines identified. In the first cycle of pregnancy, the expression of transgenic c-rel mRNA was observed, and levels of c-Rel protein were increased in the mammary gland. Importantly, 31.6% of mice developed one or more mammary tumors at an average age of 19.9 months. Mammary tumors were of diverse histology and expressed increased levels of nuclear NF-kappaB. Analysis of the composition of NF-kappaB complexes in the tumors revealed aberrant nuclear expression of multiple subunits, including c-Rel, p50, p52, RelA, RelB, and the Bcl-3 protein, as observed previously in human primary breast cancers. Expression of the cancer-related NF-kappaB target genes cyclin D1, c-myc, and bcl-xl was significantly increased in grossly normal transgenic mammary glands starting the first cycle of pregnancy and increased further in mammary carcinomas compared to mammary glands from wild-type mice or virgin transgenic mice. In transient transfection analysis in untransformed breast epithelial cells, c-Rel-p52 or -p50 heterodimers either potently or modestly induced cyclin D1 promoter activity, respectively. Lastly, stable overexpression of c-Rel resulted in increased cyclin D1 and NF-kappaB p52 and p50 subunit protein levels. These results indicate for the first time that dysregulated expression of c-Rel, as observed in breast cancers, is capable of contributing to mammary tumorigenesis.     

Indirubin enhances tumor necrosis factor-induced apoptosis through modulation of nuclear factor-kappa B signaling pathway

Although indirubin is known to exhibit anti-cancer and anti-inflammatory activities, very little is known about its mechanism of action. In this study, we investigated whether indirubin mediates its effects through interference with the NF-kappaB pathway. As examined by the DNA binding of NF-kappaB, we found that indirubin suppressed tumor necrosis factor (TNF)-induced NF-kappaB activation in a dose- and time-dependent manner. Indirubin also suppressed the NF-kappaB activation induced by various inflammatory agents and carcinogens. Further studies showed that indirubin blocked the phosphorylation and degradation of IkappaB alpha through the inhibition of activation of IkappaB alpha kinase and phosphorylation and nuclear translocation of p65. NF-kappaB reporter activity induced by TNFR1, TNF receptor-associated death domain, TRAF2, TAK1, NF-kappaB-inducing kinase, and IKKbeta was inhibited by indirubin but not that induced by p65 transfection. We also found that indirubin inhibited the expression of NF-kappaB-regulated gene products involved in antiapoptosis (IAP1, IAP2, Bcl-2, Bcl-xL, and TRAF1), proliferation (cyclin D1 and c-Myc), and invasion (COX-2 and MMP-9). This correlated with enhancement of the apoptosis induced by TNF and the chemotherapeutic agent taxol in human leukemic KBM-5 cells. Indirubin also suppressed cytokine-induced cellular invasion. Overall, our results indicate that anti-cancer and anti-inflammatory activities previously assigned to indirubin may be mediated in part through the suppression of the NF-kappaB activation pathway.     

CYLD is a crucial negative regulator of innate immune response in Escherichia coli pneumonia

Bacteraemic pneumonia is a common cause of sepsis in critically ill patients today and is characterized by dysregulation of inflammation. The genetic factors predisposing to bacteraemic pneumonia are not yet fully understood. Innate immunity is pivotal for host defence against invading bacteria, and nuclear factor-kappa B (NF-kappaB) is central to bacteria-induced inflammation and immune responses. The deubiquitinating enzyme CYLD has been identified as a key negative regulator for NF-kappaB. In the present study, we investigated the role of CYLD in innate immune response in Escherichia coli pneumonia. Upon E. coli inoculation, Cyld(-/-) mice were hypersusceptible to E. coli pneumonia with higher mortality. Innate immune response to E. coli was enhanced in Cyld(-/-) cells and mice. Cyld(-/-) cells exhibited enhanced NF-kappaB activation upon E. coli inoculation, and the enhanced NF-kappaB activation by E. coli was abolished by perturbing IkappaB kinase (IKK) signalling. Furthermore, IKK inhibitor rescued Cyld(-/-) mice from lethal infection during E. coli pneumonia along with reduced inflammation. Taken together, these data showed that CYLD acts as a crucial negative regulator for E. coli pneumonia by negatively regulating NF-kappaB. These findings provide novel insight into the regulation of bacteraemic pneumonia and related diseases and may help develop novel therapeutic strategies for these diseases.     

Epstein-Barr virus latent membrane protein 1 induces expression of the epidermal growth factor receptor through effects on Bcl-3 and STAT3

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) activates multiple signaling pathways. Two regions, C-terminal-activating region 1 (CTAR1) and CTAR2, have been identified within the cytoplasmic carboxy terminal domain that activates NF-kappaB. CTAR2 activates the canonical NF-kappaB pathway, which includes p50/p65 complexes. CTAR1 can activate both the canonical and noncanonical pathways to produce multiple distinct NF-kappaB dimers, including p52/p50, p52/p65, and p50/p50. CTAR1 also uniquely upregulates the epidermal growth factor receptor (EGFR) in epithelial cells. Increased p50-Bcl-3 complexes have been detected by chromatin precipitation on the NF-kappaB consensus motifs within the egfr promoter in CTAR1-expressing epithelial cells and nasopharyngeal carcinoma cells. In this study, the mechanism responsible for the increase in Bcl-3 has been further investigated. The data indicate that LMP1-CTAR1 induces Bcl-3 mRNA and increases the nuclear translocation of both Bcl-3 and p50. LMP1-CTAR1 constitutively activates STAT3, and this activation was not due to the induction of interleukin 6 (IL-6). In LMP1-CTAR1-expressing cells, increased levels of activated STAT3 were detected by chromatin immunoprecipitation on STAT-binding sites located within both the promoter and the second intron of Bcl-3. A STAT3 inhibitor significantly reduced the activation of STAT3, as well as the CTAR1-mediated upregulation of Bcl-3 and EGFR. These data suggest that LMP1 activates distinct forms of NF-kappaB through multiple pathways. In addition to activating the canonical and noncanonical pathways, LMP1-CTAR1 constitutively activates STAT3 and increases Bcl-3. The increased nuclear Bcl-3 and p50 homodimer complexes positively regulate EGFR expression. These results indicate that LMP1 likely regulates distinct cellular genes by activating specific NF-kappaB pathways.     

Suberoylanilide hydroxamic acid potentiates apoptosis, inhibits invasion, and abolishes osteoclastogenesis by suppressing nuclear factor-kappaB activation

Because of its ability to suppress tumor cell proliferation, angiogenesis, and inflammation, the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) is currently in clinical trials. How SAHA mediates its effects is poorly understood. We found that in several human cancer cell lines, SAHA potentiated the apoptosis induced by tumor necrosis factor (TNF) and chemotherapeutic agents and inhibited TNF-induced invasion and receptor activator of NF-kappaB ligand-induced osteoclastogenesis, all of which are known to require NF-kappaB activation. These observations corresponded with the down-regulation of the expression of anti-apoptotic (IAP1, IAP2, X chromosome-linked IAP, Bcl-2, Bcl-x(L), TRAF1, FLIP, and survivin), proliferative (cyclin D1, cyclooxygenase 2, and c-Myc), and angiogenic (ICAM-1, matrix metalloproteinase-9, and vascular endothelial growth factor) gene products. Because several of these genes are regulated by NF-kappaB, we postulated that SAHA mediates its effects by modulating NF-kappaB and found that SAHA suppressed NF-kappaB activation induced by TNF, IL-1beta, okadaic acid, doxorubicin, lipopolysaccharide, H(2)O(2), phorbol myristate acetate, and cigarette smoke; the suppression was not cell type-specific because both inducible and constitutive NF-kappaB activation was inhibited. We also found that SAHA had no effect on direct binding of NF-kappaB to the DNA but inhibited sequentially the TNF-induced activation of IkappaBalpha kinase, IkappaBalpha phosphorylation, IkappaBalpha ubiquitination, IkappaBalpha degradation, p65 phosphorylation, and p65 nuclear translocation. Furthermore, SAHA inhibited the NF-kappaB-dependent reporter gene expression activated by TNF, TNFR1, TRADD, TRAF2, NF-kappaB-inducing kinase, IkappaBalpha kinase, and the p65 subunit of NF-kappaB. Overall, our results indicated that NF-kappaB and NF-kappaB-regulated gene expression inhibited by SAHA can enhance apoptosis and inhibit invasion and osteoclastogenesis.     

Honokiol potentiates apoptosis, suppresses osteoclastogenesis, and inhibits invasion through modulation of nuclear factor-kappaB activation pathway

Recent reports have indicated that honokiol can induce apoptosis, suppress tumor growth, and inhibit angiogenesis. In this report, we found that honokiol potentiated the apoptosis induced by tumor necrosis factor (TNF) and chemotherapeutic agents, suppressed TNF-induced tumor cell invasion, and inhibited RANKL-induced osteoclastogenesis, all of which are known to require nuclear factor-kappaB (NF-kappaB) activation. Honokiol suppressed NF-kappaB activation induced by a variety of inflammatory stimuli, and this suppression was not cell type specific. Further studies showed that honokiol blocked TNF-induced phosphorylation, ubiquitination, and degradation of IkappaBalpha through the inhibition of activation of IkappaBalpha kinase and of Akt. This led to suppression of the phosphorylation and nuclear translocation of p65 and NF-kappaB-dependent reporter gene expression. Magnolol, a honokiol isomer, was equally active. The expression of NF-kappaB-regulated gene products involved in antiapoptosis (IAP1, IAP2, Bcl-x(L), Bcl-2, cFLIP, TRAF1, and survivin), proliferation (cyclin D1, cyclooxygenase-2, and c-myc), invasion (matrix metalloproteinase-9 and intercellular adhesion molecule-1), and angiogenesis (vascular endothelial growth factor) were also down-regulated by honokiol. Honokiol also down-regulated NF-kappaB activation in in vivo mouse dorsal skin model. Thus, overall, our results indicate that NF-kappaB and NF-kappaB-regulated gene expression inhibited by honokiol enhances apoptosis and suppresses osteoclastogenesis and invasion.     

Evodiamine abolishes constitutive and inducible NF-kappaB activation by inhibiting IkappaBalpha kinase activation, thereby suppressing NF-kappaB-regulated antiapoptotic and metastatic gene expression, up-regulating apoptosis, and inhibiting invasion

Evodiamine, an alkaloidal component extracted from the fruit of Evodiae fructus (Evodia rutaecarpa Benth., Rutaceae), exhibits antiproliferative, antimetastatic, and apoptotic activities through a poorly defined mechanism. Because several genes that regulate cellular proliferation, carcinogenesis, metastasis, and survival are regulated by nuclear factor-kappaB (NF-kappaB), we postulated that evodiamine mediates its activity by modulating NF-kappaB activation. In the present study, we investigated the effect of evodiamine on NF-kappaB and NF-kappaB-regulated gene expression activated by various carcinogens. We demonstrate that evodiamine was a highly potent inhibitor of NF-kappaB activation, and it abrogated both inducible and constitutive NF-kappaB activation. The inhibition corresponded with the sequential suppression of IkappaBalpha kinase activity, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation, p65 nuclear translocation, and p65 acetylation. Evodiamine also inhibited tumor necrosis factor (TNF)-induced Akt activation and its association with IKK. Suppression of Akt activation was specific, because it had no effect on JNK or p38 MAPK activation. Evodiamine also inhibited the NF-kappaB-dependent reporter gene expression activated by TNF, TNFR1, TRADD, TRAF2, NIK, and IKK but not that activated by the p65 subunit of NF-kappaB. NF-kappaB-regulated gene products such as Cyclin D1, c-Myc, COX-2, MMP-9, ICAM-1, MDR1, Survivin, XIAP, IAP1, IAP2, FLIP, Bcl-2, Bcl-xL, and Bfl-1/A1 were all down-regulated by evodiamine. This down-regulation potentiated the apoptosis induced by cytokines and chemotherapeutic agents and suppressed TNF-induced invasive activity. Overall, our results indicated that evodiamine inhibits both constitutive and induced NF-kappaB activation and NF-kappaB-regulated gene expression and that this inhibition may provide a molecular basis for the ability of evodiamine to suppress proliferation, induce apoptosis, and inhibit metastasis.     

Evidence that receptor activator of nuclear factor (NF)-kappaB ligand can suppress cell proliferation and induce apoptosis through activation of a NF-kappaB-independent and TRAF6-dependent mechanism

The receptor activator of NF-kappaB ligand (RANKL), a recently identified member of the tumor necrosis factor (TNF) superfamily, has been shown to induce osteoclastogenesis and dendritic cell survival. Most members of the TNF superfamily suppress cell proliferation and induce apoptosis, but whether RANKL does so is not known. We demonstrate that treatment of monocyte RAW 264.7 cells with RANKL induces dose-dependent growth inhibition (IC50 = 10 ng/ml) as determined by dye uptake and [3H]thymidine incorporation methods. Suppression of RANKL-induced NF-kappaB activation by dominant-negative IkappaBalpha or by the NEMO-peptide had no effect on RANKL-induced cell growth inhibition. Inhibition of RANKL-induced JNK activation, however, abolished the RANKL-induced apoptosis. Suppression of interaction of RANK with TRAF6 by TRAF6-binding peptide abrogated the anti-proliferative effects of RANKL, suggesting the critical role of TRAF6. Flow cytometric analysis of cells treated with RANKL showed accumulation of cells in G0/G1 phase of the cell cycle, and this accumulation correlated with a decline in the levels of cyclin D1, cyclin D3, and cyclin E and an increase in cyclin-dependent kinase inhibitor p27 (Kip). Flow cytometric analysis showed the presence of annexin V-positive cells in cultures treated with RANKL. RANKL-induced apoptosis was further confirmed using calcein AM/ethidium homodimer-1 dye and cleavage of poly(ADP-ribose) polymerase (PARP), procaspase 3, and procaspase 9; benzyloxycarbonyl-VAD, the pancaspase inhibitor, suppressed the PARP cleavage. Thus, overall, our studies indicate that RANKL can inhibit cell proliferation and induce apoptosis through a TRAF-6-dependent but NF-kappaB-independent mechanism.