NF-κB addiction and its role in cancer: ‘one size does not fit all’

3-Formylchromone (3-FC) has been associated with anticancer potential through a mechanism yet to be elucidated. Because of the critical role of NF-κB in tumorigenesis, we investigated the effect of this agent on the NF-κB activation pathway. Whether activated by inflammatory agents (such as TNF-α and endotoxin) or tumor promoters (such as phorbol ester and okadaic acid), 3-FC suppressed NF-κB activation. It also inhibited constitutive NF-κB expressed by most tumor cells. This activity correlated with sequential inhibition of IκBα kinase (IKK) activation, IκBα phosphorylation, IκBα degradation, p65 phosphorylation, p65 nuclear translocation, and reporter gene expression. We found that 3-FC inhibited the direct binding of p65 to DNA, and this binding was reversed by a reducing agent, thus suggesting a role for the cysteine residue. Furthermore, mutation of Cys38 to Ser in p65 abolished this effect of the chromone. This result was confirmed by a docking study. 3-FC also inhibited IKK activation directly, and the reducing agent reversed this inhibition. Furthermore, mutation of Cys179 to Ala in IKK abolished the effect of the chromone. Suppression of NF-κB activation led to inhibition of anti-apoptotic (Bcl-2, Bcl-xL, survivin, and cIAP-1), proliferative (cyclin D1 and COX-2), invasive (MMP-9 and ICAM-1), and angiogenic (VEGF) gene products and sensitization of tumor cells to cytokines. Thus, this study shows that modification of cysteine residues in IKK and p65 by 3-FC leads to inhibition of the NF-κB activation pathway, suppression of anti-apoptotic gene products, and potentiation of apoptosis in tumor cells.

The role of the cancer/testis antigen CAGE in drug resistance was investigated. The drug-resistant human melanoma Malme3M (Malme3M(R)) and the human hepatic cancer cell line SNU387 (SNU387(R)) showed in vivo drug resistance and CAGE induction. Induction of CAGE resulted from decreased expression and thereby displacement of DNA methyltransferase 1(DNMT1) from CAGE promoter sequences. Various drugs induce expression of CAGE by decreasing expression of DNMT1, and hypomethylation of CAGE was correlated with the increased expression of CAGE. Down-regulation of CAGE in these cell lines decreased invasion and enhanced drug sensitivity resulting from increased apoptosis. Down-regulation of CAGE also led to decreased anchorage-independent growth. Down-regulation of CAGE led to increased expression of p53, suggesting that CAGE may act as a negative regulator of p53. Down-regulation of p53 enhanced resistance to drugs and prevented drugs from exerting apoptotic effects. In SNU387(R) cells, CAGE induced the interaction between histone deacetylase 2 (HDAC2) and Snail, which exerted a negative effect on p53 expression. Chromatin immunoprecipitation assay showed that CAGE, through interaction with HDAC2, exerted a negative effect on p53 expression in Malme3M(R) cells. These results suggest that CAGE confers drug resistance by regulating expression of p53 through HDAC2. Taken together, these results show the potential value of CAGE as a target for the development of cancer therapeutics.

BackgroundHistone deacetylase 3 (HDAC3) is overexpressed in cancers and its inhibition enhances anti-tumor chemotherapy. ZBP-89, a transcription factor, can induce pro-apoptotic Bak and reduce HDAC3 but the mechanism is unknown. Pin1, a molecular switch that determines the fate of phosphoproteins, is known to interact with HDAC3. The aim of this study was to investigate the mechanism how ZBP-89 downregulated HDAC3.MethodsIn this study, liver cells, Pin1-knockout Pin1−/− and Pin1 wild-typed Pin+/+ cells were used to explore how ZBP-89 reduced HDAC3. The overexpression of ZBP-89 was achieved by infecting cells with Ad-ZBP-89, an adenoviral construct containing ZBP-89 gene. The role of NF-κB was determined using CAY10576, MG132 and SN50, the former two being inhibitors of IκB degradation and SN50 being an inhibitor of p65/p50 translocation. A xenograft tumor model was used to confirm the in vitro data.ResultsZBP-89 reduced HDAC3, and it could form a complex with IκB and induce IκB phosphorylation to inhibit IκB. Furthermore, ZBP-89-mediated HDAC3 reduction was suppressed by IκB degradation inhibitors CAY10576 and MG132 but not by p65/p50 translocation inhibitor SN50, indicating that IκB decrease rather than the elevated activity of NF-κB contributed to HDAC3 reduction. ZBP-89-mediated HDAC3 or IκB reduction was significantly less obvious in Pin1−/− cells compared with Pin1+/+ cells. In Ad-ZBP-89-infected Pin1+/+ cancer cells, Pin1 siRNA increased HDAC3 but decreased Bak, compared with cells without ZBP-89 infection. These findings indicate that Pin1 participates in ZBP-89-mediated HDAC3 downregulation and Bak upregulation. The cell culture result was confirmed by in vivo mouse tumor model experiments.ConclusionsZBP-89 attenuates HDAC3 by increasing IκB degradation. Such attenuation is independent of NF-κB activity but partially depends on Pin1. The novel pathway identified may help generate new anti-cancer strategy by targeting HDAC3 and its related molecules.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0382-7) contains supplementary material, which is available to authorized users.

Plumbagin, derived from the medicinal plant Plumbago zeylanica, modulates cellular proliferation, carcinogenesis, and radioresistance, all known to be regulated by the activation of the transcription factor NF-kappaB, suggesting plumbagin might affect the NF-kappaB activation pathway. We found that plumbagin inhibited NF-kappaB activation induced by TNF, and other carcinogens and inflammatory stimuli (e.g. phorbol 12-myristate 13-acetate, H2O2, cigarette smoke condensate, interleukin-1beta, lipopolysaccharide, and okadaic acid). Plumbagin also suppressed the constitutive NF-kappaB activation in certain tumor cells. The suppression of NF-kappaB activation correlated with sequential inhibition of the tumor necrosis factor (TNF)-induced activation of IkappaBalpha kinase, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation, p65 nuclear translocation, and the NF-kappaB-dependent reporter gene expression activated by TNF, TNFR1, TRAF2, NIK, IKK-beta, and the p65 subunit of NF-kappaB. Plumbagin also suppressed the direct binding of nuclear p65 and recombinant p65 to the DNA, and this binding was reversed by dithiothreitol both in vitro and in vivo. However, plumbagin did not inhibit p65 binding to DNA when cells were transfected with the p65 plasmid containing cysteine 38 mutated to serine. Plumbagin down-regulated the expression of NF-kappaB-regulated anti-apoptotic (IAP1, IAP2, Bcl-2, Bcl-xL, cFLIP, Bfl-1/A1, and survivin), proliferative (cyclin D1 and COX-2), and angiogenic (matrix metalloproteinase-9 and vascular endothelial growth factor) gene products. This led to potentiation of apoptosis induced by TNF and paclitaxel and inhibited cell invasion. Overall, our results indicate that plumbagin is a potent inhibitor of the NF-kappaB activation pathway that leads to suppression of NF-kappaB-regulated gene products. This may explain its cell growth modulatory, anticarcinogenic, and radiosensitizing effects previously described.

B7-H3, a B7 homologue, is highly expressed on tumors and tumor vasculature. Although its role as a T cell inhibitor or co-stimulator is still under investigation, high expression of membranous B7-H3 in the tumor compartment is correlated with poor overall survival. NF-kB activation, standing at the intersection between inflammation and cancer, can lead to both the production of cytokines that engage the innate immune system, as well as pro-proliferative signals in cancer. B7-H3 has been shown to enhance and suppress inflammatory signaling by modulating NF-kB. However, the mechanism by which this occurs in cancer cells remains unknown. In this study, we aim to understand how B7-H3 modulates the NF-kB pathway in order to regulate incoming inflammatory signals in cancer cells. The human colon cancer cell line, HCT116, was stably transfected with a transcriptionally-coupled post-translational NF-kB luciferase reporter comprising of the inhibitor of NF-kB, IkBα, fused to firefly luciferase and placed downstream of an NF-kB responsive promoter, kB5. This construct allows for the real-time readout of the degradation of IkBα and NF-kB driven transcriptional re-synthesis of IkBα in response to a stimulus. The reporter cell line was treated with two stimulants, TNFα and flagellin, that activate NF-kB through different pathways and to different degrees with or without treatment with human B7-H3. Treatment of the reporter cell line with B7-H3 alone did not induce IkBα degradation or re-synthesis. However, stimulation of the reporter cell line with TNFα or flagellin in combination with B7-H3 attenuated the degradation of IkBα and its re-synthesis compared to stimulation with TNFα or flagellin alone. Maximal re-synthesis of IkBα when stimulated with TNFα was 8.25 ± 1.51 fold (S.D.) and significantly increased compared to cells additionally treated with B7-H3 (5.83 ± 1.23 fold, p = .01). In addition, peak degradation time of IkBα when cells were stimulated by TNFα was 45 ± 5 min (S.D.), and significantly shorter than when cells were also treated with B7-H3 (61.67 ± 2.89 min, p = .01). Similar trends were observed when cells were treated with both flagellin and B7-H3 compared to stimulation with flagellin alone. These studies show that B7-H3 suppresses the response of colon cancer cells to incoming inflammatory stimuli and attenuates NF-kB signaling by tempering the kinetics and dynamics of IkBα. Citation Format: Sarah Glazer, Seth Gammon, David Piwnica-Worms. B7-H3 regulates NF-kB signaling in cancer cells by modulating the degradation and re-synthesis of IkBα [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5461.

Survivin is an oncogenic protein that is highly expressed in breast cancer and has a dual function that is dependent on its subcellular localization. In the cytosol, survivin blocks programmed cell death by inactivating caspase proteins; however, in the nucleus it facilitates cell division by regulating chromosomal movement and cytokinesis. In prior work, we showed that survivin is acetylated by CREB-binding protein (CBP), which restricts its localization to the nuclear compartment and thereby inhibits its anti-apoptotic function. Here, we identify histone deacetylase 6 (HDAC6) as responsible for abrogating CBP-mediated survivin acetylation in the estrogen receptor (ER)-positive breast cancer cell line, MCF-7. HDAC6 directly binds survivin, an interaction that is enhanced by CBP. In quiescent breast cancer cells in culture and in malignant tissue sections from ER+ breast tumors, HDAC6 localizes to a perinuclear region of the cell, undergoing transport to the nucleus following CBP activation where it then deacetylates survivin. Genetically modified mouse embryonic fibroblasts that lack mhdac6 localize survivin predominantly to the nuclear compartment, whereas wild-type mouse embryonic fibroblasts localize survivin to distinct cytoplasmic structures. Together, these data imply that HDAC6 deacetylates survivin to regulate its nuclear export, a feature that may provide a novel target for patients with ER+ breast cancer.

Resistance of cancer cells to powerful chemotherapeutic drugs has been an area of intense investigation. Efforts to understand the mechanisms underlying chemoresistance have led to significant progress toward our understanding of signaling pathways that favor cancer cell survival. Now that many

Post-translational modifications of RelA play an important role in regulation of NF-κB activation. We previously demonstrated that in malignant hematopoietic cells, histone deacetylase inhibitors (HDACIs) induced RelA hyperacetylation and NF-κB activation, attenuating lethality. We now present evidence that IκB kinase (IKK) β-mediated RelA Ser-536 phosphorylation plays a significant functional role in promoting RelA acetylation, inducing NF-κB activation, and limiting HDACI lethality in human multiple myeloma (MM) cells. Immunoblot profiling revealed that although basal RelA phosphorylation varied in MM cells, Ser-536 phosphorylation correlated with IKK activity. Exposure to the pan-HDACIs vorinostat or LBH-589 induced phosphorylation of IKKα/β (Ser-180/Ser-181) and RelA (Ser-536) in MM cells, including cells expressing an IκBα "super-repressor," accompanied by increased RelA nuclear translocation, acetylation, DNA binding, and transactivation activity. These events were substantially blocked by either pan-IKK or IKKβ-selective inhibitors, resulting in marked apoptosis. Consistent with these events, inhibitory peptides targeting either the NF-κB essential modulator (NEMO) binding domain for IKK complex formation or RelA phosphorylation sites also significantly increased HDACI lethality. Moreover, IKKβ knockdown by shRNA prevented Ser-536 phosphorylation and significantly enhanced HDACI susceptibility. Finally, introduction of a nonphosphorylatable RelA mutant S536A, which failed to undergo acetylation in response to HDACIs, impaired NF-κB activation and increased cell death. These findings indicate that HDACIs induce Ser-536 phosphorylation of the NF-κB subunit RelA through an IKKβ-dependent mechanism, an action that is functionally involved in activation of the cytoprotective NF-κB signaling cascade primarily through facilitation of RelA acetylation rather than nuclear translocation.

Colorectal cancer is the 3rd leading cause of cancer deaths in the United States. Activation of oncogenes and silencing of tumor suppressor genes (TSG) contribute to the development and progression of colorectal cancer. One such TSG is the transforming growth factor β receptor II (TGFβRII) which results in loss of growth inhibitory TGFβ signaling and is a common event in cancer progression. Although mutation of this TSG is common, especially in tumors which exhibit microsatellite instability there is increasing evidence that TGFβRII is epigenetically silenced in many different cancers. Restoration of TGFβ signaling by re-introduction of TGFβRII results in anti-cancer effects in colon and other cancer cell lines. Histone deacetylases (HDACs) are involved in the mechanism of epigenetic silencing. These enzymes remove the acetyl groups from lysine tails of histones usually resulting in gene repression. HDACs are classified into 4 groups (Classes I, IIa, IIb, III and IV) and, except for Class III, need the zinc cation for their function. Deacetylation by Class III HDACs (Sirtuins) is NAD+ mediated. Histone deacetylase inhibitors (HDACi) have recently entered clinical trials and are effective in inhibiting growth and inducing apoptosis in many hematological malignancies but results against solid tumors as single therapies has been disappointing. A significant effect of these drugs is reactivation of TSGs via histone deacetylation inhibition which results in alterations in the chromatin permitting transcription of these silenced genes. Our laboratory has previously demonstrated that the pan HDACi Belinostat, which inhibits Class I and II HDACs, effectively induced TGFβRII expression in cancer cell lines with epigenetically silenced receptor restoring TGFβ downstream signaling effects including the TGFβ dependent decrease in survivin. Therefore, HDACi provide a potential therapy to restore the growth inhibitory and apoptotic effects of the TGFβ inhibitory pathway. We hypothesize that the identification of the specific HDACs involved in reactivation of epigenetically silenced TGFβRII would allow the use of more specific HDACis which would increase the therapeutic index of these drugs, decrease side effects and permit more effective use in combination therapies. We are using both genetic and pharmacological approaches to identify the specific HDACs involved in reactivation of TGFβRII. We have performed lentiviral shRNA knockdown of HDAC1, 2 and 3 and observed that KD of HDACs 1 and 3 caused the induction of TGFβRII. However knock down of HDAC2 had no effect on TGFβRII expression. Treatment of colon cancer cells which exhibit epigenetically silenced TGFβRII with the HDACi Mocetinostat, specific for the Class I HDACs1, 2, 3 and 11, resulted in robust TGFβRII expression. However, treatment with Droxinostat, specific for HDACs 6 and 8, and at higher concentrations HDAC3, did not result in TGFβRII induction until concentrations effective in inhibiting HDAC3 were achieved. We are examining the effect of HDAC shRNAs and pharmacological HDAC inhibitors on XIAP and survivin expression. We are also performing Chromatin Immunoprecipitation (ChIP) to confirm that HDACs 1 and 3 the important HDACs involved in epigenetic silencing of the TGFβRII gene and whether they interact within the same transcriptional repressor complex on the promoter or exert heir effects by repressing different repressors interacting with the TGFβRII promoter. Citation Format: Catherine Murari, Gillian M. Howell, Michael G. Brattain. Identification of the specific histone deacetylases involved in the silencing of transforming growth factor β receptor II in colon cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B60.

Suberoylanilide hydroxamic acid (SAHA) is a promising histone deacetylase (HDAC) inhibitor approved by the US Food and Drug Administration (FDA) and whose clinical application for solid tumours is partially limited by decreased susceptibility in cancer cells due to nuclear factor (NF)-κB activation. As an NF-κB inhibitor, celastrol exhibits potent anticancer effects but has failed to enter clinical trials due to its toxicity. In this report, we demonstrated that the combination of celastrol and SAHA exerted substantial synergistic efficacy against human cancer cells in vitro and in vivo accompanied by enhanced caspase-mediated apoptosis. This drug combination inhibited the activation of NF-κB caused by SAHA monotherapy and consequently led to increased apoptosis in cancer cells. Interestingly, E-cadherin was dramatically downregulated in celastrol-resistant cancer cells, and E-cadherin expression was closely related to decreased sensitivity to celastrol. However, our combination treatment significantly augmented the expression of E-cadherin, suggesting that mutual mechanisms contributed to the synergistic anticancer activity. Furthermore, the enhanced anticancer efficacy of celastrol combined with SAHA was validated in a human lung cancer 95-D xenograft model without increased toxicity. Taken together, our data demonstrated the synergistic anticancer effects of celastrol and SAHA due to their reciprocal sensitisation, which was simultaneously regulated by NF-κB and E-cadherin; thus, the combination of celastrol and SAHA was superior to other combination regimens that rely on a single mechanism. Our findings not only open new opportunities for the clinical development of SAHA but should also motivate the clinical investigation of celastrol, which has been hampered by its toxicity.

Background:Over the last decade, several drugs that inhibit class I and/or class II histone deacetylases (HDACs) have been identified, including trichostatin A, the cyclic depsipeptide FR901228 and the antibiotic apicidin. These compounds have had immediate application in cancer research because of their ability to reactivate aberrantly silenced tumour suppressor genes and/or block tumour cell growth. Although a number of HDAC inhibitors are being evaluated in preclinical cancer models and in clinical trials, little is known about the differences in their specific mechanism of action and about the unique determinants of cancer cell sensitivity to each of these inhibitors.Methods:Using a combination of cell viability assays, HDAC enzyme activity measurements, western blots for histone modifications, microarray gene expression analysis and qRT–PCR, we have characterised differences in trichostatin A vs depsipeptide-induced phenotypes in lung cancer, breast cancer and skin cancer cells and in normal cells and have then expanded these studies to other HDAC inhibitors.Results:Cell viability profiles across panels of lung cancer, breast cancer and melanoma cell lines showed distinct sensitivities to the pan-inhibitor TSA compared with the class 1 selective inhibitor depsipeptide. In several instances, the cell lines most sensitive to one inhibitor were most resistant to the other inhibitor, demonstrating these drugs act on at least some non-overlapping cellular targets. These differences were not explained by the HDAC selectivity of these inhibitors alone since apicidin, which is a class 1 selective compound similar to depsipeptide, also showed a unique drug sensitivity profile of its own. TSA had greater specificity for cancer vs normal cells compared with other HDAC inhibitors. In addition, at concentrations that blocked cancer cell viability, TSA effectively inhibited purified recombinant HDACs 1, 2 and 5 and moderately inhibited HDAC8, while depsipeptide did not inhibit the activity of purified HDACs in vitro but did in cellular extracts, suggesting a potentially indirect action of this drug. Although both depsipeptide and TSA increased levels of histone acetylation in cancer cells, only depsipeptide decreased global levels of transcriptionally repressive histone methylation marks. Analysis of gene expression profiles of an isogenic cell line pair that showed discrepant sensitivity to depsipeptide, suggested that resistance to this inhibitor may be mediated by increased expression of multidrug resistance genes triggered by exposure to chemotherapy as was confirmed by verapamil studies.Conclusion:Although generally thought to have similar activities, the HDAC modulators trichostatin A and depsipeptide demonstrated distinct phenotypes in the inhibition of cancer cell viability and of HDAC activity, in their selectivity for cancer vs normal cells, and in their effects on histone modifications. These differences in mode of action may bear on the future therapeutic and research application of these inhibitors.

Two parallel interleukin-1 (IL-1)-mediated signaling pathways have been uncovered for IL-1R-TLR-mediated NFkappaB activation: TAK1-dependent and MEKK3-dependent pathways, respectively. The TAK1-dependent pathway leads to IKKalpha/beta phosphorylation and IKKbeta activation, resulting in classic NFkappaB activation through IkappaBalpha phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKgamma phosphorylation and IKKalpha activation, resulting in NFkappaB activation through dissociation of phosphorylated IkappaBalpha from NFkappaB without IkappaBalpha degradation. IL-1 receptor-associated kinase 4 (IRAK4) belongs to the IRAK family of proteins and plays a critical role in IL-1R/TLR-mediated signaling. IRAK4 kinase-inactive mutant failed to mediate the IL-1R-TLR-induced TAK1-dependent NFkappaB activation pathway, but mediated IL-1-induced TAK1-independent NFkappaB activation and retained the ability to activate substantial gene expression, indicating a structural role of IRAK4 in mediating this alternative NFkappaB activation pathway. Deletion analysis of IRAK4 indicates the essential structural role of the IRAK4 death domain in receptor proximal signaling for mediating IL-1R-TLR-induced NFkappaB activation.

miR-589-5p Inhibits Cell Proliferation by Targeting Histone Deacetylase 3 in Triple Negative Breast Cancer

Nuclear factor kappaB (NF-kappaB) has been studied extensively as an inducible transcriptional regulator of the immune and inflammatory response. NF-kappaB activation downstream of lipopolysaccharide or cytokine stimulation is controlled by the IkappaB kinase complex, which contains IKKalpha and IKKbeta. Significantly, the constitutive activity of NF-kappaB has been implicated as an important aspect of many cancer cells, but mechanisms associated with this activity are poorly understood. An inducible kinase, IKK-i/IKKepsilon, related to the catalytic forms of the IkappaB kinase, has been studied as an anti-viral, innate immune regulator through its ability to control the activity of the transcription factors IRF-3 and IRF-7. Here, we demonstrate that IKK-i/IKKepsilon is expressed in a number of cancer cells and is involved in regulating NF-kappaB activity through its ability to control basal/constitutive, but not cytokine-induced, p65/RelA phosphorylation at Ser-536, a modification proposed to contribute to the transactivation function of NF-kappaB. Knockdown of IKK-i/IKKepsilon or expression of a S536A mutant form of p65 suppresses HeLa cell proliferation. The data indicate a role for IKK-i/IKKepsilon in controlling proliferation of certain cancer cells through regulation of constitutive NF-kappaB activity.

HDAC2 promotes the migration and invasion of non-small cell lung cancer cells via upregulation of fibronectin.