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Formation of an IKKα-Dependent Transcription Complex Is Required for Estrogen Receptor-Mediated Gene Activation
Abstract
The IkappaB kinases IKKalpha and IKKbeta regulate distinct cytoplasmic and nuclear events that are critical for cytokine-mediated activation of the NF-kappaB pathway. Because the IKKs have previously been demonstrated to associate with the nuclear hormone receptor coactivator AIB1/SRC-3, the question of whether either IKKalpha or IKKbeta may be involved in increasing the expression of hormone-responsive genes was addressed. We demonstrated that IKKalpha, in conjunction with ERalpha and AIB1/SRC-3, is important in activating the transcription of estrogen-responsive genes, including cyclin D1 and c-myc, to result in the enhanced proliferation of breast cancer cells. Estrogen treatment facilitated the association of IKKalpha, ERalpha, and AIB1/SRC-3 to estrogen-responsive promoters and increased IKKalpha phosphorylation of ERalpha, AIB1/SRC-3, and histone H3. These results suggest that IKKalpha plays a major role in regulating the biological effects of estrogen via its promoter association and modification of components of the transcription complex.
... On the contrary, ERα activation by E2 via an IKK-dependent mechanism mediates SDC1 repression [98]. Mechanistically, IKKα regulates estrogen-induced phosphorylation of ERα on Ser188, which subsequently enhances ERα stability and transcriptional activity, leading to the synthesis of repressors that mediate SDC1 downregulation [98][99][100]. Interestingly, the silencing of ERβ in MDA-MB-231 breast cancer cells induces the expression of SDC1, 2, and 4 [101]. ...
... On the contrary, ERα activation by E2 via an IKK-depen mechanism mediates SDC1 repression [98]. Mechanistically, IKKα regulates estroge duced phosphorylation of ERα on Ser188, which subsequently enhances ERα stabilit transcriptional activity, leading to the synthesis of repressors that mediate SDC1 d regulation [98][99][100]. Interestingly, the silencing of ERβ in MDA-MB-231 breast cance induces the expression of SDC1, 2, and 4 [101]. ...
Simple Summary
Syndecans (SDCs; SDC1 to 4) are integral cell surface proteoglycans that are expressed normally on various types of mammalian tissues. In cancer, SDCs’ expression is dysregulated, impacting the onset and progression of cancer by modulating pivotal signaling pathways involved in biological and molecular functions. In this review, we highlight the key roles of SDCs in breast cancer pathogenesis, addressing the prognostic value and the critical molecular regulators of SDC expression, as well as different SDC-centered therapeutic perspectives.
Abstract
Syndecans (SDC1 to 4), a family of cell surface heparan sulfate proteoglycans, are frequently expressed in mammalian tissues. SDCs are aberrantly expressed either on tumor or stromal cells, influencing cancer initiation and progression through their pleiotropic role in different signaling pathways relevant to proliferation, cell-matrix adhesion, migration, invasion, metastasis, cancer stemness, and angiogenesis. In this review, we discuss the key roles of SDCs in the pathogenesis of breast cancer, the most common malignancy in females worldwide, focusing on the prognostic significance and molecular regulators of SDC expression and localization in either breast tumor tissue or its microenvironmental cells and the SDC-dependent epithelial–mesenchymal transition program. This review also highlights the molecular mechanisms underlying the roles of SDCs in regulating breast cancer cell behavior via modulation of nuclear hormone receptor signaling, microRNA expression, and exosome biogenesis and functions, as well as summarizing the potential of SDCs as promising candidate targets for therapeutic strategies against breast cancer.
... Malignant chemo-resistance is a phenomenon of cells or tissues adapting to damaging chemicals. Most first-line chemotherapeutic drugs induce a bypass effect, thereby weakening the efficacy of the drug, ultimately leading to treatment failure and tumor recurrence [4]. Activation of the NF-κB pathway is an important factor in cisplatin (CDDP) resistance [5]. ...
... Nuclear NF-κB activates anti-apoptotic genes, cell growth factors, multi-drug resistance genes, angiogenesis-related genes, and genes related to cell adhesion and metastasis, etc. at the transcriptional level, which leads to uncontrolled proliferation, apoptosis escape, and chemo-resistance of malignant cancer cells [39][40][41][42][43]. Therefore, as a potential target for treatment of malignant tumors, NF-κB is related to the prognosis of cancer [44]. However, many first-line conventional chemotherapeutic drugs, including cisplatin, always induce the activation of bypass effectors, e.g., NF-κB and snail, which weakens drug efficacy and eventually leads to treatment failure and tumor recurrence [4,6,45]. Currently, the inhibitors of NF-κB mainly include IKKs activity inhibitors, IκB phosphorylation inhibitors, and NF-κB transcription complex inhibitors, etc. ...
Wentilactone A (WA) is a tetranorditerpenoid isolated from marine algae. We previously found that WA inhibited cancer cell proliferation with little toxicity. In this study, we show that high expression of extracellular matrix protein-1 (ECM1) promotes cancer cell cisplatin resistance, and the secreted ECM1 activates normal fibroblasts (NFs) to transform cells with characteristics of cancer-associated fibroblasts (CAFs). Transcription of the ECM1 gene is regulated largely by NF-κB through EP881C/T-EP266C binding sites. WA supresses the phosphorylation of NF-κB through inhibition of the upstream IKK/IκB phoshorylation to block the expression of ECM1, which reverses the cisplatin-induced activation of NF-κB/ECM1. On the contrary, cisplatin facilitates phosphorylation of NF-κB to enhance the expression of ECM1. These results highlight ECM1 as a potential target for treatment of cisplatin-resistant cancers associated with the ECM1 activated signaling. In addition, WA reverses cisplatin resistance by targeting both tumor cells and the tumor microenvironment through IKK/IκB/NF-κB signaling to reduce the expression of the ECM1 protein.
... All experimental mice had IKKβ F/F LDLR −/− double-mutant background, and IKKβ ΔMye LDLR −/− mice carried heterozygous knock-in for LysM-Cre. All experimental mice used in this study were male, partially due to the known crosstalk between NF-κB and estrogen signaling [40,41]. However, the authors are aware of the fact that studying a single sex has limitations since sex differences have been widely reported in mouse atherosclerosis studies [42]. ...
... Nevertheless, since deficiency of myeloid IKKβ did not affect Tat-induced triglyceride levels in our study, the decreased atherosclerosis in IKKβ ΔMye LDLR −/− mice is likely due to ameliorated macrophage functions. All experimental mice used in our atherosclerosis study were male, partially due to the known crosstalk between NF-κB and estrogen signaling [40,41]. However, this is a limitation of our study since sex differences have been widely reported in mouse atherosclerosis studies [42]. ...
PurposeHIV infection is consistently associated with an increased risk of atherosclerotic cardiovascular disease, but the underlying mechanisms remain elusive. HIV protein Tat, a transcriptional activator of HIV, has been shown to activate NF-κB signaling and promote inflammation in vitro. However, the atherogenic effects of HIV Tat have not been investigated in vivo. Macrophages are one of the major cell types involved in the initiation and progression of atherosclerosis. We and others have previously revealed the important role of IκB kinase β (IKKβ), a central inflammatory coordinator through activating NF-κB, in the regulation of macrophage functions and atherogenesis. This study investigated the impact of HIV Tat exposure on macrophage functions and atherogenesis.Methods
To investigate the effects of Tat on macrophage IKKβ activation and atherosclerosis development in vivo, myeloid-specific IKKβ-deficient LDLR-deficient (IKKβΔMyeLDLR−/−) mice and their control littermates (IKKβF/FLDLR−/−) were exposed to recombinant HIV protein Tat.ResultsExposure to Tat significantly increased atherosclerotic lesion size and plaque vulnerability in IKKβF/FLDLR−/− but not IKKβΔMyeLDLR−/− mice. Deficiency of myeloid IKKβ attenuated Tat-elicited macrophage inflammatory responses and atherosclerotic lesional inflammation in IKKβΔMyeLDLR−/− mice. Further, RNAseq analysis demonstrated that HIV protein Tat affects the expression of many atherosclerosis-related genes in vitro in an IKKβ-dependent manner.Conclusions
Our findings reveal atherogenic effects of HIV protein Tat in vivo and demonstrate a pivotal role of myeloid IKKβ in Tat-driven atherogenesis.
... NF-KB directly interacts with the DNA-binding function of ER through several mechanisms, such as collaboration with FOXA1 to enhance latent ER-binding sites, and induce translation of their synergistic genes (Franco et al. 2015). In addition, NF-KB influences ER through association with its ER co-activator or co-repressor, which will then alter ER transcriptional activity (Park et al. 2005). Similar to ER, NF-KB has been reported to have a role as a downstream effector for the growth factor pathway, which is recognized to be involved in both ligand-dependent and non-ligand-dependent ER activation, leading to resistance to a wide range of antioestrogen drugs (Zhou et al. 2005a, Sas et al. 2012, Frasor et al. 2015. ...
... As previously described, oestrogen induces the formation of the IKKα, ER and steroid receptor co-activator (SRC) 3 complex on the promoter region of CYCLIN D1 gene. IKKα is also able to phosphorylate both ER and SRC3 to further increase their transcription activity (Park et al. 2005, Park 2017). In addition, IKKα has been reported as a key kinase in response to oestrogen-induced E2F1 expression. ...
Breast cancer is a heterogeneous disease, which over time acquires various adaptive changes leading to more aggressive biological characteristics and development of treatment resistance. Several mechanisms of resistance have been established, however, due to the complexity of estrogen receptor (ER) signalling and its crosstalk with other signalling networks, various areas still need to be investigated. This review focuses on the role of nuclear factor kappa B (NF-κB) as a key link between inflammation and cancer and addresses its emerging role as a key player in endocrine therapy resistance. Understanding the precise mechanism of NF-κB-driven endocrine therapy resistance provides a possible opportunity for therapeutic intervention.
... This includes transcription factors distinct from the NF-κB family, for example E2F1 [30,31], β-catenin [32], CBP [33], as well as the suppressors of transcription such as the silencing mediator for retinoic acid and thyroid hormone receptor (SMRT) [34] and cell cycle regulator cyclin D1 [35]. Additional substrates also include the protein inhibitor of activated STAT1 (PIAS1) as a modulator of transcription/inflammation [36], the oestrogen (ER) [37] and androgen receptors (AR) [38] of the steroid hormone family receptor along with their associated steroid receptor co-factor (SRC)-3 [37,39,40] and Aurora kinase A [41,42] that contributes to the mitotic process. Direct modulation of the status of these proteins by IKKα has bearing on the transcription of additional regulatory proteins such as p53 [43,44] and EZH2 [44] and additional mitotic kinase Polo-like kinase (PLK) 4 [45]. ...
... This includes transcription factors distinct from the NF-κB family, for example E2F1 [30,31], β-catenin [32], CBP [33], as well as the suppressors of transcription such as the silencing mediator for retinoic acid and thyroid hormone receptor (SMRT) [34] and cell cycle regulator cyclin D1 [35]. Additional substrates also include the protein inhibitor of activated STAT1 (PIAS1) as a modulator of transcription/inflammation [36], the oestrogen (ER) [37] and androgen receptors (AR) [38] of the steroid hormone family receptor along with their associated steroid receptor co-factor (SRC)-3 [37,39,40] and Aurora kinase A [41,42] that contributes to the mitotic process. Direct modulation of the status of these proteins by IKKα has bearing on the transcription of additional regulatory proteins such as p53 [43,44] and EZH2 [44] and additional mitotic kinase Polo-like kinase (PLK) 4 [45]. ...
The cellular kinases inhibitory-κB kinase (IKK) α and Nuclear Factor-κB (NF-κB)-inducing kinase (NIK) are well recognised as key central regulators and drivers of the non-canonical NF-κB cascade and as such dictate the initiation and development of defined transcriptional responses associated with the liberation of p52-RelB and p52-p52 NF-κB dimer complexes. Whilst these kinases and downstream NF-κB complexes transduce pro-inflammatory and growth stimulating signals that contribute to major cellular processes, they also play a key role in the pathogenesis of a number of inflammatory-based conditions and diverse cancer types, which for the latter may be a result of background mutational status. IKKα and NIK, therefore, represent attractive targets for pharmacological intervention. Here, specifically in the cancer setting, we reflect on the potential pathophysiological role(s) of each of these kinases, their associated downstream signalling outcomes and the stimulatory and mutational mechanisms leading to their increased activation. We also consider the downstream coordination of transcriptional events and phenotypic outcomes illustrative of key cancer ‘Hallmarks’ that are now increasingly perceived to be due to the coordinated recruitment of both NF-κB-dependent as well as NF-κB–independent signalling. Furthermore, as these kinases regulate the transition from hormone-dependent to hormone-independent growth in defined tumour subsets, potential tumour reactivation and major cytokine and chemokine species that may have significant bearing upon tumour-stromal communication and tumour microenvironment it reiterates their potential to be drug targets. Therefore, with the emergence of small molecule kinase inhibitors targeting each of these kinases, we consider medicinal chemistry efforts to date and those evolving that may contribute to the development of viable pharmacological intervention strategies to target a variety of tumour types.
... Nevertheless, the data suggest that the addition of exemestane changes the prognostic power of IKKa. During longterm tamoxifen treatment (five years), blockade of ER leads to higher free estrogen levels, promoting the formation of an ER/ IKKa complex to enhance transcriptional activity, which results in reduced RFS after two years, as observed by Bennett et al. (16). However, if exemestane is administered following short-term tamoxifen treatment (2.5 years), estrogen levels fall and the ER/IKKa complex is released to phosphorylate ER in a ligand-independent manner, resulting in improved RFS, as observed in the current study (16). ...
... During longterm tamoxifen treatment (five years), blockade of ER leads to higher free estrogen levels, promoting the formation of an ER/ IKKa complex to enhance transcriptional activity, which results in reduced RFS after two years, as observed by Bennett et al. (16). However, if exemestane is administered following short-term tamoxifen treatment (2.5 years), estrogen levels fall and the ER/IKKa complex is released to phosphorylate ER in a ligand-independent manner, resulting in improved RFS, as observed in the current study (16). This is not observed in the exemestane monotherapy patients, as the initial formation of the ER/IKKa complex is required to improve RFS. ...
Background:
Aromatase inhibitors improve disease-free survival compared with tamoxifen in postmenopausal women with hormone receptor-positive breast cancer. The Tamoxifen and Exemestane Adjuvant Multinational (TEAM) trial compared exemestane monotherapy with sequential therapy of tamoxifen followed by exemestane. The trial failed to show a statistically significant difference between treatment arms. A robust translational program was established to investigate predictive biomarkers.
Methods:
A tissue microarray was retrospectively constructed using a subset of patient tissues (n = 4631) from the TEAM trial (n = 9766). Immunohistochemistry was performed for biomarkers, classed into three groups: MAPK pathway, NF-kappa B pathway, and estrogen receptor (ER) phosphorylation. Expression was analyzed for association with relapse-free survival (RFS) at 2.5 and 10 years and treatment regimen using Kaplan-Meier curves and log-rank analysis. All statistical tests were two-sided.
Results:
In univariate analysis, ER167 (hazard ratio [HR] = 0.71, 95% confidence interval [CI] = 0.59 to 0.85, P < .001), IKKα (HR = 0.74, 95% CI = 0.60 to 0.92, P = .005), Raf-1338 (HR = 0.64, 95% CI = 0.52 to 0.80, P < .001), and p44/42 MAPK202/204 (HR = 0.77, 95% CI = 0.64 to 0.92, P = .004) were statistically significantly associated with improved RFS at 10 years in patients receiving sequential therapy. Associations were strengthened when IKKα, Raf-1338, and ER167 were combined into a cumulative prognostic score (HR = 0.64, 95% CI = 0.52 to 0.77, P < .001). Patients with an all negative IKKα, Raf-1338, and ER167 score favored exemestane monotherapy (odds ratio = 0.56, 95% CI = 0.35 to 0.90). In multivariable analysis, the IKKα, Raf-1338, and ER167 score (P = .001) was an independent prognostic factor for RFS at 10 years in patients receiving sequential therapy.
Conclusions:
The IKKα, Raf-1338, and ER167 score is an independent predictive biomarker for lower recurrence on sequential therapy. Negative expression may further offer predictive value for exemestane monotherapy.
... Although several studies have reported that TLR9 correlates negatively with ERα [19], others reported the opposite [20]. Park et al. have reported that an IκB kinase α (IKKα)-dependent transcription complex was required for ERα-mediated gene activation [21]. Therefore, we evaluated the protein expression levels of IKKα + β, IKKα, ERα, and phosphorylated ERα (p-ERα), and found they were increased after treatment with cfDNA ( Figure 6B). ...
... Park et al. showed that activated IKKα could also lead to the enhanced phosphorylation of ERα. IKKα and ERα were recruited to the promoter region of EREregulated genes, resulting in the activation of estrogenmediated transcription [21]. ...
In breast cancer, cell-free DNA (cfDNA) has been proven to be a diagnostic and prognostic biomarker. However, there have been few studies on the origin and biological significance of cfDNA. In this study, we assessed the release pattern of cfDNA from breast cancer cell lines under different culture conditions and investigated the biological significance of cfDNA. The cfDNA concentration increased rapidly (6 h) after passage, decreased gradually, and was then maintained at a relatively stable level after 24 h. In addition, the cfDNA concentration did not correlate with the amount of apoptotic and necrotic cells. Interestingly, if more cells were in the G1 phase, more cfDNA was detected (p < 0.01) and the cfDNA concentration correlated positively with the percent of cells in the G1 phase (p < 0.05). We observed that cells could release cfDNA actively, but not exclusively, via exosomes. Furthermore, we showed that cfDNA could stimulate hormone receptor-positive breast cancer cell proliferation by activating the TLR9-NF-κB-cyclin D1 pathway. In conclusion, cfDNA is released from breast cancer mainly by active secretion, and cfDNA could stimulate proliferation of breast cancer cells.
... 5 Upon activation of the non-canonical NF-jB pathway, the NF-jB-inducing kinase (NIK) phosphorylates IKKa which in turn phosphorylates p100 to identify it for ubiquitination and targeted proteolytic processing subsequently generating p52 and allowing the liberation of active p52/RelB dimmers. 6 These complexes translocate to the nucleus and regulate transcription of a variety of genes important in apoptosis, proliferation, invasion and adaptive immunity. [7][8][9] Therefore it is not surprising that studies have demonstrated dysregulation of the non-canonical NF-jB pathway in many solid tumours. ...
... Studies from the literature demonstrate that IKKa is involved in regulation of oestrogen-dependent genes such as cyclin D1 and c-myc, resulting in increased proliferation. 6 However, future studies in a large panel of ER positive and ER negative cell lines are required to confirm this observation. Future studies should also investigate impact of tamoxifen treatment and resistance to tamoxifen in cell line studies. ...
The aim of the current study was to examine the relationship between tumour IKKα expression and breast cancer recurrence and survival. Immunohistochemistry was employed in a discovery and a validation tissue microarray to assess the association of tumour IKKα expression and clinico-pathological characteristics. Following siRNA-mediated silencing of IKKα, cell viability and apoptosis were assessed in MCF7 and MDA-MB-231 breast cancer cells. In both the discovery and validation cohorts, associations observed between IKKα and clinical outcome measures were potentiated in oestrogen receptor (ER) positive Luminal A tumours. In the discovery cohort, cytoplasmic IKKα was associated with disease-free survival (P=0.029) and recurrence-free survival on tamoxifen (P<0.001) in Luminal A tumours. Nuclear IKKα and a combination of cytoplasmic and nuclear IKKα (total tumour cell IKKα) were associated with cancer-specific survival (P=0.012 and P=0.007, respectively) and recurrence-free survival on tamoxifen (P=0.013 and P<0.001, respectively) in Luminal A tumours. In the validation cohort, cytoplasmic IKKα was associated with cancer-specific survival (P=0.023), disease-free survival (P=0.002) and recurrence-free survival on tamoxifen (P=0.009) in Luminal A tumours. Parallel experiment with breast cancer cells in vitro demonstrated the non-canonical NF-κB pathway was inducible by exposure to lymphotoxin in ER-positive MCF7 cells and not in ER-negative MDA-MB-231 cells. Reduction in IKKα expression by siRNA transfection increased levels of apoptosis and reduced cell viability in MCF7 but not in MDA-MB-231 cells. IKKα is an important determinant of poor outcome in patients with ER-positive invasive ductal breast cancer and thus may represent a potential therapeutic target. This article is protected by copyright. All rights reserved.
... Transcription activation is regulated through two distinct activation function domains, AF1 and AF2, located in the N-terminal and Ligand Binding Domain (LBD) of ERα, respectively (3). The AF2 activity is dependent on the binding of estrogen, whereas AF1 is modulated by phosphorylation, regulated by various kinases such as MAPK, CDK7, GSK3, and IKKα in response to specific stimuli (4)(5)(6)(7)(8). The diverse genomic functions of ERα are further modulated through the recruitment of several coregulator proteins (9). ...
Estrogen receptor α (ERα) drives the transcription of genes involved in breast cancer (BC) progression, relying on coregulatory protein recruitment for its transcriptional and biological activities. Mutation of ERα as well as aberrant recruitment of its regulatory proteins contribute to tumor adaptation and drug resistance. Therefore, understanding the dynamic changes in ERα protein interaction networks is crucial for elucidating drug resistance mechanisms in BC. Despite progress in studying ERα-associated proteins, capturing subcellular transient interactions remains challenging and, as a result, significant number of important interactions remain undiscovered. In this study, we employed biotinylation by antibody recognition (BAR), an innovative antibody-based proximity labeling (PL) approach, coupled with mass spectrometry to investigate the ERα proximal proteome and its changes associated with resistance to aromatase inhibition, a key therapy used in the treatment of ERα-positive BC. We show that BAR successfully detected most of the known ERα interactors and mainly identified nuclear proteins, using either an epitope tag or endogenous antibody to target ERα. We further describe the ERα proximal proteome rewiring associated with resistance applying BAR to a panel of isogenic cell lines modeling tumor adaptation in the clinic. Interestingly, we find that ERα associates with some of the canonical cofactors in resistant cells and several proximal proteome changes are due to increased expression of ERα. Resistant models also show decreased levels of estrogen-regulated genes. Sensitive and resistant cells harboring a mutation in the ERα (Y537C) revealed a similar proximal proteome. We provide an ERα proximal protein network covering several novel ERα-proximal partners. These include proteins involved in highly dynamic processes such as sumoylation and ubiquitination difficult to detect with traditional protein interaction approaches. Overall, we present BAR as an effective approach to investigate the ERα proximal proteome in a spatial context and demonstrate its application in different experimental conditions.
... This suggests that Raf 's function in the most ventral cells is not through MAPK, but could perhaps function through direct phosphorylation of IκB/Cactus 98 (Fig. 6C) as previous studies into the oncogenic properties of Raf showed that, in mammalian cell culture, Raf regulates NFκB signaling directly by binding and phosphorylating IκB 99 . Raf could act similarly to MAP3K14, also known as NIK (NF-κB inducing Kinase), which cooperates with IKKα in mammals to phosphorylate IκB, thereby activating NF-κB signal 49,50,100 . As Raf is a MAP3K, it could play a similar role in Drosophila Toll signalling, but additional investigations into the precise molecular mechanism connecting Raf and the Toll pathway will be required. ...
Proper embryonic development requires directional axes to pattern cells into embryonic structures. In Drosophila , spatially discrete expression of transcription factors determines the anterior to posterior organization of the early embryo, while the Toll and TGFβ signalling pathways determine the early dorsal to ventral pattern. Embryonic MAPK/ERK signaling contributes to both anterior to posterior patterning in the terminal regions and to dorsal to ventral patterning during oogenesis and embryonic stages. Here we describe a novel loss of function mutation in the Raf kinase gene, which leads to loss of ventral cell fates as seen through the loss of the ventral furrow, the absence of Dorsal/NFκB nuclear localization, the absence of mesoderm determinants Twist and Snail, and the expansion of TGFβ. Gene expression analysis showed cells adopting ectodermal fates much like loss of Toll signaling. Our results combine novel mutants, live imaging, optogenetics and transcriptomics to establish a novel role for Raf, that appears to be independent of the MAPK cascade, in embryonic patterning.
... Studies have shown that NF-κB directly interacts with the DNA-binding function of ER through a variety of mechanisms (Franco et al., 2015). NF-κB can also affect ER by binding to the ER co-activator or co-inhibitor, which will change the ER transcriptional activity (Park et al., 2005). In addition, NF-κB directly binds to the cyclin D1 promoter and controls the transcription of cyclin D1 (Guttridge et al., 1999). ...
HER2+/HR+ breast cancer is a special molecular type of breast cancer. Existing treatment methods are prone to resistance; “precision treatment” is necessary. Pyrotinib is a pan-her kinase inhibitor that can be used in HER2-positive tumors, while SHR6390 is a CDK4/6 inhibitor that can inhibit ER+ breast cancer cell cycle progression and cancer cell proliferation. In cancer cells, HER2 and CDK4/6 signaling pathways could be nonredundant; co-inhibition of both pathways by combination of SHR6390 and pyrotinib may have synergistic anticancer activity on HER2+/HR+ breast cancer. In this study, we determined the synergy of the two-drug combination and underlying molecular mechanisms. We showed that the combination of SHR6390 and pyrotinib synergistically inhibited the proliferation, migration, and invasion of HER2+/HR+ breast cancer cells in vitro . The combination of two drugs induced G1/S phase arrest and apoptosis in HER2+/HR+ breast cancer cell lines. The combination of two drugs prolonged the time to tumor recurrence in the xenograft model system. By second-generation RNA sequencing technology and enrichment analysis of the pyrotinib-resistant cell line, we found that FOXM1 was associated with induced resistance to HER2-targeted therapy. In HER2+/HR+ breast cancer cell lines, the combination of the two drugs could further reduce FOXM1 phosphorylation, thereby enhancing the antitumor effect to a certain extent. These findings suggest that SHR6390 combination with pyrotinib suppresses the proliferation, migration, and invasion of HER2+/HR+ breast cancers through regulation of FOXM1.
... All mice used in this study had IKKβ fl/fl background, and IKKβ ΔFib mice carried heterozygous knockin for tamoxifen-inducible Cre recombinase. All experimental mice used in this study were male littermates, partially due to the known crosstalk between NF-κB and estrogen signaling (69,70). However, the authors are aware of the fact that studying a single sex has limitations since sex differences have been widely reported in mouse studies. ...
Cardiac inflammation and fibrosis contribute significantly to hypertension-related adverse cardiac remodeling. IκB kinase β (IKKβ), a central coordinator of inflammation through activation of NF-κB, has been demonstrated as a key molecular link between inflammation and cardiovascular disease. However, the cell-specific contribution of IKKβ signaling towards adverse cardiac remodeling remains elusive. Cardiac fibroblasts are one of the most populous non-myocyte cell types in the heart that play a key role in mediating cardiac fibrosis and remodeling. To investigate the function of fibroblast IKKβ, we generated inducible fibroblast-specific IKKβ-deficient mice. Here we report an important role of IKKβ in the regulation of fibroblast functions and cardiac remodeling. Fibroblast-specific IKKβ deficient male mice were protected from angiotensin II (Ang II)-induced cardiac hypertrophy, fibrosis, and macrophage infiltration. Ablation of fibroblast IKKβ inhibited Ang II-stimulated fibroblast proinflammatory and profibrogenic responses, leading to ameliorated cardiac remodeling and improved cardiac function in IKKβ-deficient mice. Findings from this study establish fibroblast IKKβ as a key factor regulating cardiac fibrosis and function in hypertension-related cardiac remodeling.
... This suggests that Raf's function in the most ventral cells is not through MAPK, but could perhaps function through direct phosphorylation of IκB/Cactus [99]. Alternatively, Raf could act similarly to MAP3K14, also known as NIK (NF-kB inducing Kinase), which cooperates with IKKα in mammals to phosphorylate IκB, thereby activating NF-κB signal [49,50,100]. As Raf is a MAP3K, it could play a similar role in Drosophila Toll signalling, but additional investigations into the precise molecular mechanism connecting Raf and the Toll pathway will be required. ...
Proper embryonic development requires directional axes to pattern cells into embryonic structures. In Drosophila, spatially discrete expression of transcription factors determines the anterior to posterior organization of the early embryo, while the Toll and TGFβ signalling pathways determine the early dorsal to ventral pattern. Embryonic MAPK/ERK signaling contributes to both anterior to posterior patterning in the terminal regions and to dorsal to ventral patterning during oogenesis and embryonic stages. Here we describe a novel loss of function mutation in the Raf kinase gene, which leads to loss of ventral cell fates as seen through the loss of the ventral furrow, the absence of Dorsal/NFκB nuclear localization, the absence of mesoderm determinants Twist and Snail, and the expansion of TGFβ. Gene expression analysis showed cells adopting ectodermal fates much like loss of Toll signaling. Our results combine novel mutants, live imaging, optogenetics and transcriptomics to establish a novel role for Raf, that appears to be independent of the MAPK cascade, in embryonic patterning.
... CRC: colorectal cancer; IKKα: inhibitor of kappaB kinase alpha; BRAF: serine/threonine-specific protein kinase of the RAF family; TAK1: TGFβ-Activated kinase 1; SMRT: silencing-mediator for retinoid/ thyroid hormone receptors; MAPK: mitogen-activated protein kinase; ATM: ataxia telangiectasia mutated gene; NEMO: NF-κB essential modulator; PRC2: polycomb repressive complex 2; KAP1: KRAB-associated protein-1; RIF1: replication timing regulatory factor 1 IKKα is also responsible for the tumor-promoting effect of progesterone in breast cancer downstream of receptor activator of NF-κB ligand (RANKL) induction [39,40] and for the metastatic spread of breast cancer cells, which depends on RANKL produced by tumor-infiltrating regulatory T cells [41] . IKKα also phosphorylates Estrogen Receptor α, its coactivator AIB1/SRC3, and induces downstream targets such as cyclin D1 and c-myc, promoting breast cancer cell proliferation as well [42,43] . In a clinical study, Bennett et al. [44] observed that IKKα expression in breast cancer cells is associated with patient outcome independently of their cellular localization. ...
Cancer therapy has improved considerably in the last years; however, therapeutic resistance is still a major problem that impedes full response to the treatment and the main cause of patient relapse and death. Numerous kinases have been reported to be overactivated in cancer and induce resistance to current therapies. Targeting kinases has proven to be useful for overcoming chemotherapy resistance and thus improving patient outcomes. Inhibitor of kappaB kinase alpha (IKKα) is a serine/threonine kinase that was first described as part of the IKK complex in the nuclear factor-κB (NF-κB) pathway, which regulates several physiological and physiopathological processes such as immunity, inflammation, and cancer. However, the IKKα subunit has been shown to be dispensable for NF-κB activation and responsible of multiple pro-tumorigenic functions. Furthermore, we identified a nuclear active form of IKKα kinase IKKα(p45) that promotes tumor growth and therapy resistance, independent of canonical NF-κB. Improved understanding of resistance mechanisms will facilitate drug discovery and provide new effective therapies. Here, we review the recent publications on the implications of IKKα in cancer initiation, development, and resistance.
... pubs.acs.org/acsmedchemlett Letter nuclear activity of IKKα has been connected to an upregulation of NF-κB signaling 24 and has been linked to cell cycle regulation and survival in colorectal, 25,26 breast, 27,28 pancreatic, 29 gastric, 30 osteo-sarcoma, 31 and prostate cancers. 32−34 These results prompted us to synthesize a small library of gibberellin and allogibberic acid analogs based on a synthetic strategy we had previously developed toward the synthesis of pharbinilic acid, 22 with the goal of identifying structural features necessary for activity. ...
div>The small molecule gibberellin JRA-003 (1) was identified as an inhibitor of the NF-kB (nuclear kappa-light-chain enhancer of activated B cells) pathway. Here we find that JRA-003 binds to and significantly inhibits the nuclear translocation of pathway-activating kinases IKKalpha (IkB kinase alpha) and IKKbeta (IkB kinase beta). Analogs were synthesized and NF-kB-inhibiting gibberellins were found to be cytotoxic in cancer-derived cell lines (HS 578T, HCC 1599, RC-K8, Sud-HL4, CA 46, and NCIH 4466). Not only was JRA-003 (1) identified as the most potent synthetic gibberellin against cancer-derived cell lines, it displayed no cytotoxicity in cells derived from non-cancerous sources (HEK 293T, HS 578BST, HS 888Lu, HS 895Sk, HUVEC). This selectivity suggests a promising approach for the development of new therapeutics.</div
... The mechanism by which estrogens regulate cyclin D1 levels in hormone-responsive breast cancer cells is mainly transcriptional. Although no estrogen-responsive element-(ERE) -related sequence has been identified in the cyclin D1 promoter, several potential estrogenresponsive sites have been mapped in the cyclin D1 proximal promoter [24,40,[51][52][53]. ...
Background:
Androgens, through their own receptor, play a protective role on breast tumor development and progression and counterbalance estrogen-dependent growth stimuli which are intimately linked to breast carcinogenesis.
Methods:
Cell counting by trypan blu exclusion was used to study androgen effect on estrogen-dependent breast tumor growth. Quantitative Real Time RT-PCR, western blotting, transient transfection, protein immunoprecipitation and chromatin immunoprecipitation assays were carried out to investigate how androgen treatment and/or androgen receptor overexpression influences the functional interaction between the steroid receptor coactivator AIB1 and the estrogen- or androgen receptor which, in turn affects the estrogen-induced cyclin D1 gene expression in MCF-7 breast cancer cells. Data were analyzed by ANOVA.
Results:
Here we demonstrated, in estrogen receptor α (ERα)-positive breast cancer cells, an androgen-dependent mechanism through which ligand-activated androgen receptor (AR) decreases estradiol-induced cyclin D1 protein, mRNA and gene promoter activity. These effects involve the competition between AR and ERα for the interaction with the steroid receptor coactivator AIB1, a limiting factor in the functional coupling of the ERα with the cyclin D1 promoter. Indeed, AIB1 overexpression is able to reverse the down-regulatory effects exerted by AR on ERα-mediated induction of cyclin D1 promoter activity. Co-immunoprecipitation studies indicated that the preferential interaction of AIB1 with ERα or AR depends on the intracellular expression levels of the two steroid receptors. In addition, ChIP analysis evidenced that androgen administration decreased E2-induced recruitment of AIB1 on the AP-1 site containing region of the cyclin D1 gene promoter.
Conclusions:
Taken together all these data support the hypothesis that AIB1 sequestration by AR may be an effective mechanism to explain the reduction of estrogen-induced cyclin D1 gene activity. In estrogen-dependent breast cancer cell proliferation, these findings reinforce the possibility that targeting AR signalling may potentiate the effectiveness of anti-estrogen adjuvant therapies.
... Bennett et al. demonstrated that silencing the expression of IKKα only affected the proliferation of the Luminal A breast cancer cell line MCF-7 and not the Basal cell line MDA-MB-231 [58]. In agreement, Park et al. demonstrated that IKKα participates in the regulation of estrogen-genes such as cyclin D1 and c-myc [101]. ...
Inflammation is a well-known driver of carcinogenesis and cancer progression, often attributed to the tumor microenvironment. However, tumor cells themselves are capable of secreting a variety of inflammatory molecules, leading to the activation of specific signaling pathways that promote tumor progression. The NF-κB signaling pathway is one of the most important connections between inflammation and tumorigenesis. NF-κB is a superfamily of transcription factors that plays an important role in several types of hematological and solid tumors, including breast cancer. However, the role of the NF-κB pathway in the survival of breast cancer patients is poorly studied. In this study, we analyzed and related the expression of both canonical and alternative NF-κB pathways and selected target genes with the relapse-free and overall survival of breast cancer patients. We used the public database Kaplan-Meier plotter (KMplot) which includes gene expression data and survival information of 3951 breast cancer patients. We found that the expression of IKKα was associated with poor relapse-free survival in patients with ER-positive tumors. Moreover, the expression of IL-8 and MMP-1 was associated with poor relapse-free and overall survival. In contrast, expression of IKKβ, p50, and p65 from the canonical pathway, and NIK and RELB from the alternative pathway correlated with better relapse-free survival also when the patients were classified by their hormonal and nodal status. Our study suggests that the expression of genes of the canonical and alternative NF-κB pathways is ultimately critical for tumor persistence. Understanding the communication between both pathways would help to find better therapeutic and prophylactic targets to prevent breast cancer progression and relapse.
... The activity of the 26S proteasome controls the level of ERα, and the reduction of phosphorylation of ERα-Ser118 phosphorylation correlates with faster elimination of E2-induced ER and inhibition of ERαmediated gene regulation (La Rosa et al. 2012). Estrogeninduced phosphorylation of ERα and its transcriptional activation through phosphorylation of ERα on Ser118 preferentially involves IκB kinase-α (IKK-α) (Park et al. 2005, Weitsman et al. 2006). However, IKKβ and IKKϵ regulate as well, ERα activity and the expression of its target genes (Guo et al. 2016, El-Shennawy et al. 2018. ...
Breast cancer (BC) is the primary cause of cancer-related mortality among women. Patients who express the estrogen receptor (ER), which mediates the tumorigenic effects of estrogens, respond to antihormonal therapy. Loss of ER expression or acquired resistance to E2 is associated with aggressive malignant phenotypes, which lead to relapse. These BC subtypes overexpress syndecan-1 (SDC1), a transmembrane heparan sulfate proteoglycan that mediates angiogenesis as well as the proliferation and invasiveness of cancer cells. We showed here that the activation of ER-alpha (ERα) by estrogens induces downregulation of SDC1 expression in ER(+) MCF7 cells but not in T47D cells. Loss of ERα expression, induced by RNA interference or a selective ER down-regulator, led to subsequent SDC1 overexpression. E2-dependent downregulation of SDC1 expression required de novo protein synthesis and was antagonized by treatment with BAY 11-7085, an irreversible inhibitor of IκBα phosphorylation, which inhibits the activation of NFκB. Downregulation of SDC1 expression required ERα and activation of IKK, but was independent to downstream transcriptional regulators of NFκB. BAY 11-7085 prevented E2-mediated phosphorylation of ERα on Ser118, increasing its proteasomal degradation, suggesting that IKK stabilized E2-activated ERα, leading to subsequent downregulation of SDC1 expression. Our results showed that sustained ER signaling inhibits SDC1 expression. Such antagonism elucidates the inverse correlation between SDC1 and ER expression in ER(+) BC as well as the overexpression of SDC1 in hormone receptor-negative BC subtypes with the most aggressive phenotypes. These results identify SDC1 as an attractive therapeutic target for BC as well as for other endocrine-associated cancers.
... Although many features of active transcriptional enhancers have been characterized, the order of events leading to enhancer formation and activation as well as the dynamics of co-regulator interactions within the enhancer complex is yet unclear. Nevertheless, early kinetic studies using ChIP-PCR targeted at EREs in close proximity to the promoter of the estrogen responsive TFF1, cyclin D1 (CCND1), cathepsin D (CATD) and c-Myc (MYC) genes indicated that the association of ERα and Pol II with promoters shows a periodic engagement (171)(172)(173)(174). Moreover, detailed studies utilizing the transcriptionally synchronized estrogen responsive TFF1 promoter as a model through the use of α-amanitin to reversibly inhibit Pol II activity indicate that the engagement of ERα with the proximal ERE of TFF1occurs cyclically with short periods (175). ...
The effects of E2 (17beta-estradiol) on cells are primarily conveyed by the transcription factors, estrogen receptor (ER) alpha and beta. The regulation of responsive genes by the well- defined ER alpha in response to E2 relies on complex and highly organized processes that dynamically integrate functions of many transcription regulators to induce spatiotemporal alterations in chromatin state and structure. Changes in gene expressions result in cell-specific responses that include proliferation, differentiation and death. Deregulation of E2-ER alpha signaling contributes to the initiation and progression of target tissue malignancies. We aim here to provide a review of recent findings on dynamic transcriptional events mediated by E2-ER alpha with the anticipation that a better understanding of complex regulatory mechanisms underlying ER actions would be a critical basis for the development of effective prognostic tools for and therapeutic interventions against estrogen target tissue malignancies.
... IKKα ve IKKβ, NF-ß B yolundaki rollerinden bağımsız olarak meme kanseri hücrelerinde onkojenik işlevleri sergilemektedir. Östrojene tepki olarak, İKKα, artan gen transkripsiyonuna yol açan, siklin D1 ve c-myc de dâhil olmak üzere östrojen yanıtlı promoterlere östrojen reseptör alfa (ER) ve steroid reseptör koaktivatör 3'ü (SRC-3) güçlendirir [62]. Aktivasyon üzerine, İKKα bu hücrelerin çekirdeğine girer ve p27 / Kip1'i fosforile ederek çekirdek transferini tetikler bu da hücre proliferasyonunda artışa neden olur [63]. ...
... Although many features of active transcriptional enhancers have been characterized, the order of events leading to enhancer formation and activation as well as the dynamics of co-regulator interactions within the enhancer complex is yet unclear. Nevertheless, early kinetic studies using ChIP-PCR targeted at EREs in close proximity to the promoter of the estrogen responsive TFF1, cyclin D1 (CCND1), cathepsin D (CATD) and c-Myc (MYC) genes indicated that the association of ERα and Pol II with promoters shows a periodic engagement (171)(172)(173)(174). Moreover, detailed studies utilizing the transcriptionally synchronized estrogen responsive TFF1 promoter as a model through the use of α-amanitin to reversibly inhibit Pol II activity indicate that the engagement of ERα with the proximal ERE of TFF1occurs cyclically with short periods (175). ...
17beta-estradiol (E2), the main circulating estrogen hormone, is involved in a wide variety of physiological functions ranging from the development to the maintenance of many tissues and organs. The effects of E2 on cells are primarily conveyed by the transcription factors, estrogen receptor (ER) alpha and beta. The regulation of responsive genes by the well-defined ER alpha in response to E2 relies on complex and highly organized processes that dynamically integrate functions of many transcription regulators to induce spatiotemporal alterations in chromatin state and structure. Changes in gene expressions result in cell-specific responses that include proliferation, differentiation and death. Deregulation of E2-ER alpha signaling contributes to the initiation and progression of target tissue malignancies. We aim here to provide a review of recent findings on dynamic transcriptional events mediated by E2-ER alpha with the anticipation that a better understanding of complex regulatory mechanisms underlying ER actions would be a critical basis for the development of effective prognostic tools for and therapeutic interventions against estrogen target tissue malignancies.
... A functional DNA binding domain is not necessary for phosphorylation of ER at S118 in response to E2. The phosphorylation of ER at S118 is mediated by a variety of kinases, including mitogen-activated protein kinase, cyclin-dependent kinase 7 (CDK7), glycogen synthase kinase 3, and IB kinase alpha, depending on the stimulus (12,14,39,42,43). Additionally, various studies have shown that ER is capable of being phosphorylated by the DNA-dependent protein kinase (DNA-PK) holoenzyme (44)(45)(46). ...
Post-translational modifications are key regulators of protein function, providing cues that can alter protein interactions and cellular location. Phosphorylation of the estrogen receptor (ER) at serine 118 (pS118-ER) occurs in response to multiple stimuli and is involved in modulating ER-dependent gene transcription. While the cistrome of ER is well established, surprisingly little is understood about how phosphorylation impacts ER-DNA binding activity. To define the pS118-ER cistrome, ChIP-seq was performed on pS118-ER and ER in MCF-7 cells treated with estrogen. pS118-ER occupied a subset of ER binding sites which were associated with the active enhancer mark, acetylated H3K27. Unlike ER, pS118-ER sites were enriched in GRHL2 DNA binding motifs, and estrogen treatment increased GRHL2 recruitment to sites occupied by pS118-ER. Additionally, pS118-ER occupancy sites showed greater enrichment of full length estrogen response elements relative to ER sites. In an in vitro DNA binding array of genomic binding sites, pS118-ER was more commonly associated with direct DNA binding events compared to indirect binding events. These results indicate that phosphorylation of ER at serine 118 promotes direct DNA binding at active enhancers and is a distinguishing mark for associated transcription factor complexes on chromatin.
... Quantitative re-analysis of independent studies Given we found a robust and stable response to ER activation by estra-2-diol in contrast to the cyclical response previously described [1], we reviewed studies that have investigated ER binding at the TFF1 promoter. Several studies either used a different promoter [24], factor [25] or estra-2-diol concentration/include α-amanitin [2]. ...
Estrogen Receptor-alpha (ER) is the key driver of 75% of all breast cancers. Upon stimulation by its ligand estra-2-diol, ER forms a transcriptionally active complex binding chromatin. Previous studies have reported that ER binding follows a cyclical binding pattern with a periodicity of 90 minutes. However, these studies have been limited to individual ER target genes and most were done without replicates. Thus, the robustness and generality of ER cycling are not well understood.
Here we present a comprehensive genome-wide analysis of the time dependence of ER binding affinity up to 90 minutes after activation, based on 6 replicates at 10 time points using our previously reported method for precise quantification of binding, Parallel-Factor ChIP-seq (pfChIP-seq). In contrast to previously described cyclical binding, our approach identifies a unidirectional sustained increase in ER binding affinity, as well as a class of estra-2-diol independent binding sites. Our results are corroborated by a quantitative re-analysis of data from multiple independent studies.
Our new model reconciles the results of multiple conflicting studies into the activation of ER at the TFF1 promoter. We provide a detailed understanding of ER's response to estra-2-diol in the context of the receptor's fundamental role as both the main driver and therapeutic target of breast cancer.
... Ce résidu sérine peut également être phosphorylé par le facteur de transcription TFIIH (transcription factor II H) qui contient la kinase CDK7 dont l'activité est dépendante du ligand et du domaine AF-2 (Chen et al., 2000). La kinase IKKα (IκB kinase α) activée de façon ligand dépendante est indispensable à la phosphorylation de cette sérine (Park et al., 2005). L'ubiquitinylation des ER induit leur dégradation par le protéasome (Nawaz et al., 1999). ...
Le cancer du sein est le cancer le plus fréquent chez la femme. Sa croissance et sa progression sont dépendantes de signaux hormonaux, tels que les œstrogènes, et de l’interaction des cellules cancéreuses avec le microenvironnement matriciel. La perte d’expression du récepteur aux estrogènes ERα est un marqueur de mauvais pronostic et de non réponse à l’hormonothérapie. Dans ces tumeurs agressives, l’expression du SDC-1, un protéoglycane transmembranaire impliqué dans l’angiogenèse, la prolifération et l’invasion, est augmentée suggérant un antagonisme entre la signalisation œstrogénique et le SDC-1 dans ces tumeurs. En utilisant les cellules de carcinome mammaire ER(+) MCF7, nous avons montré que les œstrogènes via le récepteur ERα sont capables d’inhiber l’expression du SDC 1 par un mécanisme nécessitant une néosynthèse protéique et la phosphorylation d’ERα par la kinase IKK. Parallèlement, nos résultats montrent que la dérégulation de l’expression du SDC-1 affecte la réponse proliférative des œstrogènes dans les cellules MCF7 en modifiant la localisation subcellulaire d’ERα. Nos résultats montrent à la fois l’inhibition tonique et E2-induite de l’expression du SDC-1 par ERα dans les cellules MCF7 et la potentialité du SDC-1 à réduire la réponse œstrogénique de ces cellules. Ainsi, ces résultats sont autant d’arguments qui renforcent l'hypothèse d'un antagonisme entre la signalisation médiée par ERα et le SDC-1 lequel influencerait l'orientation phénotypique des cellules de carcinome mammaire.
... NF-κB's function as a tumor promoter is also due to its role in driving cell proliferation and protecting cells from cell death under stress conditions by regulating the expression and activity of target genes involved in cell cycle progression and apoptosis [5][6][7]9,49,[86][87][88]. Canonical NF-κB was shown to activate genes involved in cell cycle progression such as CcnD1 [5,86,89,90], E2F1 [5,86], and several E2F target genes [5] and the mitotic checkpoint Ser/Thr-protein kinase BUB1 [34]. It was also shown to suppress genes involved in apoptosis such as FOXO3a, leading to increased cell survival [4,21,91,92]. ...
The NF-κB family of transcription factors regulate the expression of genes encoding proteins and microRNAs (miRNA, miR) precursors that may either positively or negatively regulate a variety of biological processes such as cell cycle progression, cell survival, and cell differentiation. The NF-κB-miRNA transcriptional regulatory network has been implicated in the regulation of proinflammatory, immune, and stress-like responses. Gene regulation by miRNAs has emerged as an additional epigenetic mechanism at the post-transcriptional level. The expression of miRNAs can be regulated by specific transcription factors (TFs), including the NF-κB TF family, and vice versa. The interplay between TFs and miRNAs creates positive or negative feedback loops and also regulatory networks, which can control cell fate. In the current review, we discuss the impact of NF-κB-miRNA interplay and feedback loops and networks impacting on inflammation in cancer. We provide several paradigms of specific NF-κB-miRNA networks that can regulate inflammation linked to cancer. For example, the NF-κB-miR-146 and NF-κB-miR-155 networks fine-tune the activity, intensity, and duration of inflammation, while the NF-κB-miR-21 and NF-κB-miR-181b-1 amplifying loops link inflammation to cancer; and p53- or NF-κB-regulated miRNAs interconnect these pathways and may shift the balance to cancer development or tumor suppression. The availability of genomic data may be useful to verify and find novel interactions, and provide a catalogue of 162 miRNAs targeting and 40 miRNAs possibly regulated by NF-κB. We propose that studying active TF-miRNA transcriptional regulatory networks such as NF-κB-miRNA networks in specific cancer types can contribute to our further understanding of the regulatory interplay between inflammation and cancer, and also perhaps lead to the development of pharmacologically novel therapeutic approaches to combat cancer.
... We now know that primary human breast cancer specimens and breast cancer cell lines exhibit aberrant IKKa and IKKb kinase activities that may not involve NF-jB [95] or its downstream targets [96] to enhance transformation of breast cancer cells. IKKa/b kinases have been also shown to promote mammary tumorigenesis through inhibition of FOXO3a [97] and glycogen synthase kinase 3b by the activation of mTORC2, which further regulates the phosphatidylinositol-3-kinase-AKT pathway [98]. Moreover, implication of a role for constitutively active IKK complex and NF-jB activation in breast cancer cell migration and metastasis has also been reported [99]. ...
NF-κB transcription factors are the central hubs of the signaling pathways that connect pro-inflammatory signaling to cell survival, proliferation and cytokine production. In cancers, NF-κB signaling influences many aspects of tumor development, from initiation to metastasis. These functions are mediated by tumor-induced plasticity that allows tumor cells to adapt and survive the evolving conditions within the tumor microenvironment. Tumor cell plasticity is shaped by the inflammatory microenvironment in tumors. Our review focuses on IκB kinases, the direct upstream elements of NF-κB regulation, specifically on their conventional and non-conventional functions in animal models of tumorigenesis from the recent literature. This article is protected by copyright. All rights reserved.
... The most detailed ChIP-based analysis of the dynamic cofactors recruitment has been obtained for ERαmediated gene expression on pS2/TFF1, cyclin D1, cathepsin D (CATD) and c-Myc promoters (Shang et al, 2000;Reid et al, 2003;Métivier et al, 2003 ;Liu & Bagchi, 2004;Park et al, 2005). These studies indicate that the recruitment of coregulators follows a precise timed and ordered pattern. ...
Thyroid hormone (T3) controls both developmental and physiological processes. Its nuclear receptors, thyroid hormone receptors (TRs), are members of the nuclear hormone receptor family which act as ligand-dependent transcription factors. DNA methylation at the fifth position of cytosine is an important epigenetic modification that affects chromatin structure and gene expression. Recent studies have established a critical function of the Ten-eleven translocation (TET) family proteins in regulating DNA methylation dynamics by converting 5-methyl-cytosine (5mC) into 5-hydroxymethylcytosine (5hmC). Studies demonstrated that TETs proteins (including TET1, TET2 and TET3) possess catalytic activity dependent and independent transcriptional regulatory functions. Our study identified TET3 as a new TR interacting protein. The AF2 domain of TR and the catalytic domain and CXXC domain of TET3 are responsible for their interaction. This interaction allows the stabilization of chromatin bound TR, resulting in a potentiation of its transcriptional activity. The modulation effect of TET3 on TR presented here is independent of its hydroxylase activity. Thus this study evidences a new mode of action for TET3 as a non-classical regulator of TR, modulating its stability and access to chromatin rather that its intrinsic transcriptional activity. Mutations in TR cause the RTH symptom which severity varies with the particular mutation. The differential ability of different TRα mutants, relevant for the human RTHα disease, to interact with TET3 might explain their differential dominant negative activity. The regulatory function of TET3 might be more general towards the nuclear receptor transcriptional factors since different members of the superfamily present the same interaction with TET3, such as AR (androgen receptor), ERR (Estrogen-related receptor) and RAR (retinoic acid receptor). The interaction between TET3 and RAR involves the DNA binding domain of RAR. The functional relevance of TET3/RAR interaction was further studied in ES cells. Combined deficiency of all three TETs led to depletion of 5hmC and deregulation of genes involved in ES differentiation. Among the deregulated genes, a subset of RA response genes was identified, suggesting that RARs (retinoic acid receptors) and TETs might work together to regulate ES cell differentiation. Further dissection revealed that TET proteins may have a role in facilitating RAR recruitment to the promoter regions of these RAR target genes. Moreover, our results indicated a potential role of the hydroxylase activity of TET proteins in modulating RAR transcriptional activity. Altogether, our work identified TET proteins as new regulators of NR (Nuclear Receptors). The exact mechanisms involved need to be further studied.
... 3,4,6 IKKα has been shown to have a minor role in the canonical pathway 4,6 but is pivotal in the noncanonical pathway, catalyzing the phosphorylation and proteolytic processing of p100 NF-κB2 which in turn liberates distinct NF-κB p52/RelB dimers and initiates transcription of a specific subset of genes. IKKα and IKKβ have specific cellular functions, 3,8,9 and the selective inhibition of one isoform over the other may provide a useful and novel therapeutic strategy in cancer and inflammatory diseases. ...
IKKβ plays a central role in the canonical NF-kB pathway, which has been extensively characterised. The role of IKKα in the non-canonical NF-kB pathway, and indeed in the canonical pathway as a complex with IKKβ, is less well understood. One major reason for this is the absence of chemical tools designed as selective inhibitors for IKKα over IKKβ, until now. Herein, we report for the first time a series of novel, potent and selective inhibitors of IKKα. We demonstrate effective target engagement and selectivity with IKKα in U2OS cells through inhibition of IKKα-driven p100 phosphorylation in the non-canonical NF-kB pathway without affecting IKKβ-dependent IKappa-Bα loss in the canonical pathway. These compounds represent the first chemical tools that can be used to further characterise the role of IKKα in cellular signalling, to dissect this from IKKβ and validate it in its own right as a target in inflammatory diseases.
... In response to estrogen, IKKα increases phosphorylation and recruitment of estrogen receptor alpha (ERα) and steroid receptor coactivator 3 (SRC-3) to estrogen-responsive promoters, including cyclin D1 and c-myc, leading to enhanced gene transcription. Activation of these genes increases estrogen-dependent proliferation of breast cancer cells [20]. IKKα can also cooperate with Notch-1 to induce the transcriptional activation of ERα-dependent genes [21]. ...
Nuclear factor-κB (NF-κB) has been long considered a master regulator of inflammation and immune responses. Additionally, aberrant NF-κB signaling has been linked with carcinogenesis in many types of cancer. In recent years, the study of NF-κB members in NF-κB unrelated pathways provided novel attractive targets for cancer therapy, specifically linked to particular pathologic responses. Here we review specific functions of IκB kinase complexes (IKKs) and IκBs, which have distinctly tumor promoting or suppressing activities in cancer. Understanding how these proteins are regulated in a tumor-related context will provide new opportunities for drug development.
... Consistent with selectivity for the canonical pathway, no impact on the noncanonical gene target Cyclin D1 was observed (Supplementary Figure 4). [33][34][35][36][37][38] In summary, we implemented a Synthetic Loop Replacement (SLR) strategy to develop an inhibitor against the NEMO-IKKα/β interaction. Utilizing this strategy, we demonstrated that installation of a hydrocarbon bridge can be used to stabilize the IKK loop structure, a feature necessary for interaction with NEMO. ...
Aberrant canonical NF-κB signaling is implicated in diseases from autoimmune disorders to cancer. A major therapeutic challenge is the need for selective inhibition of the canonical pathway without impacting the many non-canonical NF-κB functions. Here we show that a selective peptide-based inhibitor of canonical NF-κB signaling, in which a hydrogen bond in the NBD peptide is synthetically replaced by a non-labile bond, shows an about 10-fold increased potency relative to the original inhibitor. Not only is this molecule, NBD2, a powerful tool for dissection of canonical NF-κB signaling in disease models and healthy tissues, the success of the synthetic loop replacement suggests that the general strategy could be useful for discovering modulators of the many protein-protein interactions mediated by such structures.
... We also present a new model proposing that cyclin-dependent recruitment of CDK2 to the structurally activated domain E (LBD) of ERα enables pS294 formation ( Figure 5). While other AF-1 domain phosphorylation events have been shown to occur by kinase recruitment via receptor coactivators like AIB1/ SRC-3 [32] or by LBD residues that can directly dock to a specific kinase [33], our findings are the first to suggest that hinge phosphorylation at S294 first depends ...
ERα phosphorylation at hinge site S294 (pS294) was recently shown to be essential for ER-dependent gene transcription and mediated by an unknown cyclin-dependent kinase (CDK). This study was undertaken to identify the exact CDK pathway mediating pS294 formation, and to determine if this phosphorylation event occurs with, and can be targeted to treat, the ligand-independent growth of breast cancers expressing endocrine-refractory ESR1 mutations. Using a newly developed anti-pS294 monoclonal antibody, a combination of CDK specific siRNA knockdown studies and a broad panel of CDK selective inhibitors against ligand (E2)-stimulated MCF7 cells, we first identified CDK2 as the primary mediator of pS294 formation and showed that CDK2-selective inhibitors like Dinaciclib, but not CDK4/6 inhibitors like Palbociclib, can selectively prevent pS294 formation and repress ER-dependent gene expression. We then expressed the ER-activating mutations ERmut(Y537S) and ERmut(D538G) in MCF7 cells, and demonstrated their ability to induce ligand-independent and tamoxifen-resistant growth, associated with constitutive and CDK2-dependent pS294 expression. Following robust growth of E2-independent and TAM-resistant MCF7mutER(Y537S) tumors in vivo, nude mice were also treated with either Dinaciclib or Palbociclib at doses and injection schedules unable to retard tumor growth as single agents; the TAM plus Palbociclib combination arrested further tumor growth without affecting pS294 formation, while the TAM plus Dinaciclib combination produced tumor regression associated with loss of pS294 expression. These findings, and our proposed mechanistic model, provide new rationale for the clinical evaluation of CDK2 inhibitors given in combination with endocrine agents as a new treatment strategy against ESR1 mutation expressing breast cancers.
... IKKα is a component of the IKK complex. 5 In addition to its cytoplasmic functions where it controls the activation of both classical and alternative NF-κB pathways, 6,[9][10][11][12] IKKα can function as a nuclear protein kinase, [33][34][35] and nuclear IKKα plays important roles in prostate cancer progression and metastasis. 13,14 In the present study we have shown that IKKα controls the biogenesis of a group of miRNA, including miR-196a. ...
Radioresistance is a major obstacle in successful clinical cancer radiotherapy, and the underlying mechanisms are not clear. Here we show that IKKα-mediated miR-196a biogenesis via interaction with Drosha regulates the sensitivity of nasopharyngeal carcinoma (NPC) cells to radiotherapy. Phosphorylation of IKKα at T23 site (p-IKKαT23) promotes the binding of IKKα to Drosha that accelerates the processing of miR-196a primary transcripts, leading to increased expressions of both precursor and mature miR-196a. Dephosphorylation of p-IKKαT23 downregulates miR-196a expression and promotes the resistance of NPC cells to radiation treatment. The miR-196a mimic suppresses while its inhibitor promotes the resistance of NPC to radiation treatment. Importantly, the expression of p-IKKαT23 is positively related to the expression of miR-196a in human NPC tissues, and expression of p-IKKαT23 and miR-196a is inversely correlated with NPC clinical radioresistance. Thus, our studies establish a novel mechanistic link between the inactivation of IKKαT23-Drosha-miR-196a pathway and NPC radioresistance, and de-inactivation of IKKαT23-Drosha-miR-196a pathway would be an efficient way to restore the sensitivity of radioresistant NPC to radiotherapy.Cell Death and Differentiation advance online publication, 8 April 2016; doi:10.1038/cdd.2016.32.
Endocrine therapy that blocks estrogen signaling is the most effective treatment for patients with estrogen receptor positive (ER+) breast cancer. However, the efficacy of agents such as tamoxifen (Tam) is often compromised by the development of resistance. Here we report that cytokines-activated nuclear IKKα confers Tam resistance to ER+ breast cancer by inducing the expression of FAT10, and that the expression of FAT10 and nuclear IKKα in primary ER+ human breast cancer was correlated with lymphotoxin β (LTB) expression and significantly associated with relapse and metastasis in patients treated with adjuvant mono-Tam. IKKα activation or enforced FAT10 expression promotes Tam-resistance while loss of IKKα or FAT10 augments Tam sensitivity. The induction of FAT10 by IKKα is mediated by the transcription factor Pax5, and coordinated via an IKKα-p53-miR-23a circuit in which activation of IKKα attenuates p53-directed repression of FAT10. Thus, our findings establish IKKα-to-FAT10 pathway as a new therapeutic target for the treatment of Tam-resistant ER+ breast cancer.
NF-κB signaling has been discovered for nearly 40 years. Initially, NF-κB signaling was identified as a pivotal pathway in mediating inflammatory responses. However, with extensive and in-depth investigations, researchers have discovered that its role can be expanded to a variety of signaling mechanisms, biological processes, human diseases, and treatment options. In this review, we first scrutinize the research process of NF-κB signaling, and summarize the composition, activation, and regulatory mechanism of NF-κB signaling. We investigate the interaction of NF-κB signaling with other important pathways, including PI3K/AKT, MAPK, JAK-STAT, TGF-β, Wnt, Notch, Hedgehog, and TLR signaling. The physiological and pathological states of NF-κB signaling, as well as its intricate involvement in inflammation, immune regulation, and tumor microenvironment, are also explicated. Additionally, we illustrate how NF-κB signaling is involved in a variety of human diseases, including cancers, inflammatory and autoimmune diseases, cardiovascular diseases, metabolic diseases, neurological diseases, and COVID-19. Further, we discuss the therapeutic approaches targeting NF-κB signaling, including IKK inhibitors, monoclonal antibodies, proteasome inhibitors, nuclear translocation inhibitors, DNA binding inhibitors, TKIs, non-coding RNAs, immunotherapy, and CAR-T. Finally, we provide an outlook for research in the field of NF-κB signaling. We hope to present a stereoscopic, comprehensive NF-κB signaling that will inform future research and clinical practice.
Osteoarthritis (OA) is the most prevalent disorder of synovial joint affecting multiple joints. In the past decade, we have witnessed conceptual switch of OA pathogenesis from a ‘wear and tear’ disease to a disease affecting entire joint. Extensive studies have been conducted to understand the underlying mechanisms of OA using genetic mouse models and ex vivo joint tissues derived from individuals with OA. These studies revealed that multiple signalling pathways are involved in OA development, including the canonical Wnt/β‐catenin signalling and its interaction with other signalling pathways, such as transforming growth factor β (TGF‐β), bone morphogenic protein (BMP), Indian Hedgehog (Ihh), nuclear factor κB (NF‐κB), fibroblast growth factor (FGF), and Notch. The identification of signalling interaction and underlying mechanisms are currently underway and the specific molecule(s) and key signalling pathway(s) playing a decisive role in OA development need to be evaluated. This review will focus on recent progresses in understanding of the critical role of Wnt/β‐catenin signalling in OA pathogenesis and interaction of β‐catenin with other pathways, such as TGF‐β, BMP, Notch, Ihh, NF‐κB, and FGF. Understanding of these novel insights into the interaction of β‐catenin with other pathways and its integration into a complex gene regulatory network during OA development will help us identify the key signalling pathway of OA pathogenesis leading to the discovery of novel therapeutic strategies for OA intervention.
Introduction:
Despite the advances made in cancer treatment in the past decades, therapeutic efficacy is still quite challenging, partially due to the emergence of multidrug resistance (MDR). It is crucial to decipher the underlying mechanisms of resistance in order to develop new therapeutic strategies for cancer patients. Previous studies have shown that activation of nuclear factor-κB (NF-κB) plays key roles in various cellular processes including proliferation, anti-apoptosis, metastasis, invasion, and chemoresistance.
Areas covered:
In this review, we conduct an integrated analysis of the evidence suggesting the vital roles of the NFκB signaling pathway in MDR during chemotherapy, immunotherapy, endocrine and targeted therapy. A literature search was performed on NFκB and drug resistance in PubMed up to Feb 2023.
Expert opinion:
This review summarizes that the NF-κB signaling pathway exhibits a crucial role in enhancing drug resistance in chemotherapy, immunotherapy, endocrine and targeted therapy. The application of combination therapy with existing antineoplastic drugs and a safe NF-κB inhibitor could become a promising strategy in cancer treatment. A better understanding of the pathway and mechanisms of drug resistance may help exploit safer and more effective NF-κB-targeting agents for clinical use in the future.
Asthmatic women tend to develop severe airway disease in their reproductive years, and 30%–40% of asthmatic women have peri-menstrual worsening of asthma symptoms. This indicates that fluctuations in ovarian hormones are involved in advancement of asthmatic disease and exacerbation of symptoms. Group 2 innate lymphoid cells, or ILC2, are readily detected in allergic conditions, such as rhinosinusitis, in individuals that develop nasal polyps do to allergen exposures, and in allergic asthma. ILC2 are airway localized immune cells activated by IL-33, an innate cytokine that perpetuates allergic inflammation by driving the production of IL-5 and IL-13. We have previously shown that ILC2 are highly activated in naïve and ovalbumin (OVA) challenged, female BALB/c mice in comparison to male mice following stimulation with IL-33. Here, we investigated the effect of steady-state ovarian hormones on ILC2 and the NF-κB signaling pathway following OVA sensitization and challenge. We found that estrogen-treated ovariectomized mice (OVX-E2) that had been challenged with OVA had reduced IL-5 and IL-13 production by lung ILC2 as compared to lung ILC2 isolated from intact male and female sham-operated controls that had been treated with OVA. ILC2 were isolated from untreated animals and co-cultured ex vivo with and without estrogen plus IL-33. Those estrogen-treated ILC2 similarly produced less IL-5 and IL-13 in comparison to untreated, and had reduced NF-κB activation. Single-cell RNA sequencing showed that 120 genes were differentially expressed in male and female ILC2, and Nfkb1 was found among top-ranked regulatory interactions. Together, these results provide new insight into the suppressive effect of estrogen on ILC2 which may be protective in female asthmatics. Understanding further how estrogen modulates ILC2 may provide therapeutic targets for the treatment of allergic diseases.
The IKK family is most well studied in the context of control of inflammatory gene expression, particularly in the regulation of NF-κB and IRF transcription factors.
While the major targets of IKK in regulation of NF-κB and IRF signaling have been extensively studied, IKK family members are now known to phosphorylate proteins not related to NF-κB- or IRF-dependent transcription mechanisms. Recently, a variety of novel IKK family-dependent phosphorylation events have been described. This aspect of IKK function, in our opinion, is currently underappreciated and is critical for understanding the biology of these kinases and understanding effects of IKK family-directed inhibitors.
The nuances of these regulatory mechanisms are especially important since aberrant regulation of IKK family kinases has been implicated in a wide range of human diseases, including cancer, obesity, diabetes, heart disease, and pathogen infection.
The inhibitor of kappa B kinase (IKK) family consists of IKKα, IKKβ, and the IKK-related kinases TBK1 and IKKε. These kinases are considered master regulators of inflammation and innate immunity via their control of the transcription factors NF-κB, IRF3, and IRF7. Novel phosphorylated substrates have been attributed to these kinases, a subset of which is not directly related to either inflammation or innate immunity. These findings have greatly expanded the perspectives on the biological activities of these kinases. In this review we highlight some of the novel substrates for this kinase family and discuss the biological implications of these phosphorylation events.
The small molecule gibberellin JRA-003 (1) was identified as an inhibitor of the NF-κB (nuclear kappa-light-chain-enhancer of activated B cells) pathway. Here we find that JRA-003 (1) binds to and significantly inhibits the nuclear translocation of pathway-activating kinases IKKα (IΚB kinase alpha) and IKKβ (IΚB kinase beta). Analogs of 1 were synthesized and NF-κB-inhibiting gibberellins were found to be cytotoxic in cancer-derived cell lines (HS 578T, HCC 1599, RC-K8, Sud-HL4, CA 46, and NCIH 4466). Not only was JRA-003 (1) identified as the most potent synthetic gibberellin against cancer-derived cell lines, it displayed no cytotoxicity in cells derived from non-cancerous sources (HEK 293T, HS 578BST, HS 888Lu, HS 895Sk, HUVEC). This selectivity suggests a promising approach for the development of new therapeutics.
Estrogen Receptor-alpha (ER) drives 75% of breast cancers. Stimulation of the ER by estra-2-diol forms a transcriptionally-active chromatin-bound complex. Previous studies reported that ER binding follows a cyclical pattern. However, most studies have been limited to individual ER target genes and without replicates. Thus, the robustness and generality of ER cycling are not well understood. We present a comprehensive genome-wide analysis of the ER after activation, based on 6 replicates at 10 time-points, using our method for precise quantification of binding, Parallel-Factor ChIP-seq. In contrast to previous studies, we identified a sustained increase in affinity, alongside a class of estra-2-diol independent binding sites. Our results are corroborated by quantitative re-analysis of multiple independent studies. Our new model reconciles the conflicting studies into the ER at the TFF1 promoter and provides a detailed understanding in the context of the ER's role as both the driver and therapeutic target of breast cancer.
Recent studies have shown that progesterone receptor (PR)-expressing cells respond to progesterone in part through the induction of the receptor activator of NF-kappaB ligand (RANKL) in which acts in a paracrine manner to induce expansion of a RANK-expressing luminal progenitor cell population. The RANK+ population in human breast tissue from carriers of BRCA1 mutations (BRCA1mut/+) as well as the luminal progenitor population in Brca1-deficient mouse mammary glands is abnormally amplified. Remarkably, mouse Brca1+/- and human BRCA1mut/+ progenitor cells are able to form colonies in vitro in the absence of progesterone demonstrating a hormone-independent proliferative capacity. Our research has demonstrated that proliferation in BRCA1-deficient cells results in a DNA damage response (DDR) that activates a persistent NF-kappaB signal which supplants progesterone/RANKL signaling for an extended time period. Thus, the transcriptional targets normally activated by RANKL that promote a proliferative response in luminal progenitors can contribute to the susceptibility of mammary epithelial cells to BRCA1-mutated breast cancers as a consequence of DDR-induced NF-kappaB. Together these latest findings mark substantial progress in uncovering the mechanisms driving high rates of breast tumorigenesis in BRCA1 mutation carriers and ultimately reveal possibilities for non-surgical prevention strategies.
Aberrant canonical NF-κB signaling is implicated in diseases from autoimmune disorders to cancer. A major therapeutic challenge is the need for selective inhibition of the canonical pathway without impacting the many non-canonical NF-κB functions. Here we show that a selective peptide-based inhibitor of canonical NF-κB signaling, in which a hydrogen bond in the NBD peptide is synthetically replaced by a non-labile bond, shows an about 10-fold increased potency relative to the original inhibitor. Not only is this molecule, NBD2, a powerful tool for dissection of canonical NF-κB signaling in disease models and healthy tissues, the success of the synthetic loop replacement suggests that the general strategy could be useful for discovering modulators of the many protein–protein interactions mediated by such structures.
Sustained estrogenic exposure increases the risk and/or the progression of various cancers, including those of the breast, endometrium and ovary. Unexpectedly, physiological level of estrogen together with a novel IKKα inhibitor BAY11-7082 could effectively induce cell apoptosis in ER-positive breast cancer cells, suggesting combining estrogen with IKKα inhibition may be beneficial for breast cancer patients. This opinion article touches upon the dual role estrogen played in inducing cancer cell death and asks whether use of estrogen in combination with IKKα-targeted therapy would be possible reconsider the newly identified crosstalk between ER and NFκB pathway which can be utilized to switch the effects of estrogen on cell death.
Nonmelanoma skin cancers (NMSC) are the most common human malignancies. IKKα is an essential protein for skin development and is also involved in the genesis and progression of NMSC, through mechanisms not fully understood. While different studies show that IKKα protects against skin cancer, others indicate that it promotes NMSC. To resolve this controversy we have generated two models of transgenic mice expressing the IKKα protein in the nucleus (N-IKKα mice) or the cytoplasm (C-IKKα mice) of keratinocytes. Chemical skin carcinogenesis experiments show that tumors developed by both types of transgenic mice exhibit histological and molecular characteristics that make them more prone to progression and invasion than those developed by Control mice. However, the mechanisms through which IKKα promotes skin tumors are different depending on its subcellular localization; while IKKα of cytoplasmic localization increases EGFR, MMP-9 and VEGF-A activities in tumors, nuclear IKKα causes tumor progression through regulation of c-Myc, Maspin and Integrin-α6 expression. Additionally, we have found that N-IKKα skin tumors mimic the characteristics associated to aggressive human skin tumors with high risk to metastasize. Our results show that IKKα has different non-overlapping roles in the nucleus or cytoplasm of keratinocytes, and provide new targets for intervention in human NMSC progression.
Aromatase inhibitors (AIs) target the production of estrogens with the intention of reducing estrogen receptor signalling in breast cancer. One mechanism by which tumour cells can evade AI therapy is to find alternative ways to activate the estrogen receptor in the absence of ligand. Here we discuss mechanisms of ligand-independent receptor activation including growth factor cross talk, kinase induced phosphorylation, the involvement of co-factors as well as hypersensitivity of the estrogen receptor. Understanding how cell signalling pathways regulate estrogen receptor activity is helping to identify biomarkers of AI resistance. The research discussed here has also led to the development of a number of new treatment strategies to help combat AI resistant disease.
Steroid receptor coactivators (SRCs), including SRC-1, SRC-2, and SRC-3, mediate transcriptional activities of nuclear receptors and other transcription factors. SRCs’ activities and functions are regulated by multiple signaling pathways, including those of hormones, growth factors, and cytokines, and are determined by post-translational modifications, including phosphorylation, ubiquitination, sumoylation, acetylation, and methylation. SRCs integrate signals from a variety of pathways that regulate multiple cellular processes such as metabolism, reproduction, and growth. For the growth response, they regulate proliferation, survival, migration, and invasion, and promote tumor development and metastasis. SRCs are highly disregulated in many types of cancers at multiple levels including gene amplification, mutation, and mRNA/protein overexpression. Alterations of SRCs are frequently associated with advanced tumor progression and drug resistance. As such, SRCs are important prognostic cancer biomarkers and could serve as therapeutic targets for cancer therapy.
Ganoderma lucidum, an oriental medical mushroom, has been used in Asia for the prevention and treatment of a variety of diseases, including cancer. We have previously demonstrated that G. lucidum inhibits growth and induces cell cycle arrest at G(0)/G(1) phase through the inhibition of Akt/NF-kappa B signaling in estrogen-independent human breast cancer cells. However, the molecular mechanism(s) responsible for the inhibitory effects of G. lucidum on the proliferation of estrogen-dependent (MCF-7) and estrogen-independent (MDA-MB-231) breast cancer cells remain to be elucidated. Here, we show that G. lucidum inhibited the proliferation of breast cancer MCF-7 and MDA-MB-231 cells by the modulation of the estrogen receptor (ER) and NF-kappa B signaling. Thus, G. lucidum down-regulated the expression of ER alpha in MCF-7 cells but did not effect the expression of ER beta in MCF-7 and MDA-MB-231 cells. In addition, G. lucidum inhibited estrogen-dependent as well as constitutive transactivation activity of ER through estrogen response element (ERE) in a reporter gene assay. G. lucidum decreased TNF-alpha-induced (MCF-7) as well as constitutive (MDA-MB-231) activity of NF-kappa B. The inhibition of ER and NF-kappa B pathways resulted in the down-regulation of expression of c-myc, finally suppressing proliferation of estrogen-dependent as well as estrogen-independent cancer cells. Collectively, these results suggest that G. lucidum inhibits proliferation of human breast cancer cells and contain biologically active compounds with specificity against estrogen receptor and NF-kappa B signaling, and implicate G. lucidum as a suitable herb for chemoprevention and chemotherapy of breast cancer.
The advent of genome-wide transcription factor profiling has revolutionized the field of breast cancer research. Estrogen receptor alpha (ERα), the major drug target in hormone receptor-positive breast cancer, has been known as a key transcriptional regulator in tumor progression for over 30 years. Even though this function of ERα is heavily exploited and widely accepted as an Achilles heel for hormonal breast cancer, only since the last decade we are beginning to understand how this transcription factor is functioning on a genome-wide scale. Initial ChIP-on-chip analyses have taught us that ERα is an enhancer-associated factor binding to many thousands of sites throughout the human genome, and revealed the identity of a number of directly interacting transcription factors that are essential for ERα action. More recently, with the development of massive parallel sequencing technologies and refinements thereof in sample processing, a genome-wide interrogation of ERα has become feasible and affordable with unprecedented data quality and richness. These studies have revealed numerous additional biological insights in ERα behaviour in cell lines and especially in clinical specimens. So what have we actually learned during this first decade of cistromics in breast cancer and where may future developments in the field take us?
The transcription factor NF-kappaB has been the focus of intense investigation for nearly two decades. Over this period, considerable progress has been made in determining the function and regulation of NF-kappaB, although there are nuances in this important signaling pathway that still remain to be understood. The challenge now is to reconcile the regulatory complexity in this pathway with the complexity of responses in which NF-kappaB family members play important roles. In this review, we provide an overview of established NF-kappaB signaling pathways with focus on the current state of research into the mechanisms that regulate IKK activation and NF-kappaB transcriptional activity.
Phosphorylation of the estrogen receptor α (ERα) N-terminal transcription activation function AF1 at serine 118 (S118) modulates its activity. We show here that human ERα is phosphorylated by the TFIIH cyclin-dependent kinase in a ligand-dependent manner. Furthermore, the efficient phosphorylation of S118 requires a ligand-regulated interaction of TFIIH with AF2, the activation function located in the ligand binding domain (LBD) of ERα. This interaction involves (1) the integrity of helix 12 of the LBD/AF2 and (2) p62 and XPD, two subunits of the core TFIIH. These findings are suggestive of a novel mechanism by which nuclear receptor activity can be regulated by ligand-dependent recruitment of modifying activities, such as kinases.
The estrogen receptor (ER) is an important regulator of growth and differentiation of breast epithelium. Transactivation by ER depends on a leucine-rich motif, which constitutes a ligand-regulated binding site for steroid receptor coactivators (SRCs). Cyclin D1 is frequently amplified in breast cancer and can activate ER through direct binding. We show here that cyclin D1 also interacts in a ligand-independent fashion with coactivators of the SRC-1 family through a motif that resembles the leucine-rich coactivator binding motif of nuclear receptors. By acting as a bridging factor between ER and SRCs, cyclin D1 can recruit SRC-family coactivators to ER in the absence of ligand. A cyclin D1 mutant that binds to ER but fails to recruit coactivators preferentially interferes with ER activation in breast cancer cells that have high levels of cyclin D1. These data support that cyclin D1 contributes significantly to ER activation in breast cancers in which the protein is overexpressed. Our present results reveal a novel route of coactivator recruitment to ER and establish a direct role for cyclin D1 in regulation of transcription.
IkappaB kinases (IKKs) IKK1 and IKK2 are two putative IkappaBalpha kinases involved in NF-kappaB activation. To examine the in vivo functions of IKK1, we generated IKK1-deficient mice. The mutant mice are perinatally lethal and exhibit a wide range of developmental defects. Newborn mutant mice have shiny, taut, and sticky skin without whiskers. Histological analysis shows thicker epidermis, which is unable to differentiate. Limbs and tail are wrapped inside the skin and do not extend properly out of the body trunk. Skeleton staining reveals a cleft secondary palate, split sternebra 6, and deformed incisors. NF-kappaB activation mediated by TNFalpha and IL-1 is diminished in IKK1-deficient mouse embryonic fibroblast (MEF) cells. The IKK complex in the absence of IKK1 is capable of phosphorylating IkappaBalpha and IkappaBbeta in vitro. Our results support a role for IKK1 in NF-kappaB activation and uncover its involvement in skin and skeleton development. We conclude further that the two related kinases IKK1 and IKK2 have distinct functions and can not be substituted for each other's functions.
17β-Estradiol (E2) induces expression of several genes via estrogen receptor (ER)-Sp1 protein interactions with GC-rich promoter
elements in which Sp1 but not ER binds DNA. This study reports the ligand- and cell context-dependent ERα/Sp1 and ERβ/Sp1 action using an E2-responsive construct (pSp1) containing a GC-rich promoter. Both ERα and ERβ proteins physically interact with Sp1 (coimmunoprecipitation) and preferentially bind to the C-terminal region of this protein
in pull-down assays. E2- and antiestrogen-dependent transcriptional activation of ERα/Sp1 was observed in MCF-7, MDA-MB-231, and LnCaP cells, but not in HeLa cells. E2 did not affect or significantly decrease
ERβ/Sp1 action, and antiestrogens had minimal effects in the same 4 cell lines. Exchange of activation function-1 (AF-1) domains
of ER subtypes gave chimeric ERα/β(AF-1α/AF-2β) and ERβ/α (AF-1β/AF-2α) proteins that resembled wild-type ER (α or β) in terms of physical association with Sp1 protein. Transcriptional
activation studies with chimeric ERβ/α and ERα/β showed that only ERα/β can activate transcription from an Sp1 element, not ERβ/α. This indicates that the AF-1 domain from ERα is responsible for activation at an Sp1 element, independent of ER subtype context. In order to further characterize this
observation, deletion constructs in the AF-1 domain of both ERα and ERα/β were made, and transactivation studies indicated that the region between amino acids 79 and 117 of this domain is important
for activation at an Sp1 element.
Growth factor modulation of estrogen receptor (ER) activity plays an important role in both normal estrogen physiology and
the pathogenesis of breast cancer. Growth factors are known to stimulate the ligand-independent activity of ER through the
activation of mitogen-activated protein kinase (MAPK) and the direct phosphorylation of ER. We found that the transcriptional
activity of AIB1, a ligand-dependent ER coactivator and a gene amplified preferentially in ER-positive breast cancers, is
enhanced by MAPK phosphorylation. We demonstrate that AIB1 is a phosphoprotein in vivo and can be phosphorylated in vitro
by MAPK. Finally, we observed that MAPK activation of AIB1 stimulates the recruitment of p300 and associated histone acetyltransferase
activity. These results suggest that the ability of growth factors to modulate estrogen action may be mediated through MAPK
activation of the nuclear receptor coactivator AIB1.
Our appreciation of the physiological functions of estrogens and the mechanisms through which estrogens bring about these functions has changed during the past decade. Just as transgenic mice were produced in which estrogen receptors had been inactivated and we thought that we were about to understand the role of estrogen receptors in physiology and pathology, it was found that there was not one but two distinct and functional estrogen receptors, now called ER alpha and ER beta. Transgenic mice in which each of the receptors or both the receptors are inactive have revealed a much broader role for estrogens in the body than was previously thought. This decade also saw the description of a male patient who had no functional ER alpha and whose continued bone growth clearly revealed an important function of estrogen in men. The importance of estrogen in both males and females was also demonstrated in the laboratory in transgenic mice in which the aromatase gene was inactivated. Finally, crystal structures of the estrogen receptors with agonists and antagonists have revealed much about how ligand binding influences receptor conformation and how this conformation influences interaction of the receptor with coactivators or corepressors and hence determines cellular response to ligands.
We aimed to investigate the dynamics of the NF-kappaB signaling pathway in living cells using GFP variants of p65-NF-kappaB, IkappaBalpha, tumor necrosis factor-receptor associated factor 2 (TRAF2), the NF-kappaB inducing kinase (NIK) and IkappaB kinases (IKK1 and IKK2). Detailed kinetic analysis of constitutive nucleocytoplasmic shuttling processes revealed that IkappaBalpha enters the nucleus faster than p65. Examination of signaling molecules upstream of NF-kappaB and IkappaBalpha revealed a predominant cytoplasmic localization at steady state. However, after addition of leptomycin B, NIK rapidly accumulated in the nucleus, whereas we could not detect any significant effect on TRAF2 or IKK2. Using various truncation mutants of NIK, we identified a functional nuclear export signal within the COOH-terminal region 795-805, which counteracts the inherent NLS at amino acids 143-149. Prolonged incubation in the presence of LMB also leads to nuclear accumulation of IKK1, which was dependent on a lysine residue at position 44, which is also essential for kinase activity. Investigation of endogenous protein levels by immunofluorescence staining and Western blots verified the results obtained with GFP chimeras. We conclude that NF-kappaB.IkappaB complexes and the upstream signaling kinases NIK and IKK1 shuttle between cytoplasm and nucleus of nonactivated cells and that this process leads to a basal transcriptional activity of NF-kappaB.
In the past few years, many nuclear receptor coactivators have been identified and shown to be an integral part of receptor action. The most frequently studied of these coactivators are members of the steroid receptor coactivator (SRC) family, SRC-1, TIF2/GRIP1/SRC-2, and pCIP/ACTR/AIB-1/RAC-3/TRAM-1/SRC-3. In this report, we describe the biochemical purification of SRC-1 and SRC-3 protein complexes and the subsequent identification of their associated proteins by mass spectrometry. Surprisingly, we found association of SRC-3, but not SRC-1, with the I kappa B kinase (IKK). IKK is known to be responsible for the degradation of I kappa B and the subsequent activation of NF-kappa B. Since NF-kappa B plays a key role in host immunity and inflammatory responses, we therefore investigated the significance of the SRC-3-IKK complex. We demonstrated that SRC-3 was able to enhance NF-kappa B-mediated gene expression in concert with IKK. In addition, we showed that SRC-3 was phosphorylated by the IKK complex in vitro. Furthermore, elevated SRC-3 phosphorylation in vivo and translocation of SRC-3 from cytoplasm to nucleus in response to tumor necrosis factor alpha occurred in cells, suggesting control of subcellular localization of SRC-3 by phosphorylation. Finally, the hypothesis that SRC-3 is involved in NF-kappa B-mediated gene expression is further supported by the reduced expression of interferon regulatory factor 1, a well-known NF-kappa B target gene, in the spleens of SRC-3 null mutant mice. Taken together, our results not only reveal the IKK-mediated phosphorylation of SRC-3 to be a regulated event that plays an important role but also substantiate the role of SRC-3 in multiple signaling pathways.
IkappaB kinase-alpha (IKK-alpha) exhibits protein-kinase-dependent and -independent functions. Its kinase activity is required for lymphoid organogenesis and mammary gland development, whereas a kinase-independent activity is required for epidermal keratinocyte differentiation. In addition to failed epidermal differentiation, IKK-alpha-deficient mice exhibit abnormal skeletal and craniofacial morphogenesis. As similar defects are not exhibited by mice that experience systemic inhibition of NF-kappaB, we postulated that the morphogenetic defects in IKK-alpha-deficient mice are not caused by reduced NF-kappaB activity but instead are due to failed epidermal differentiation that disrupts proper epidermal-mesodermal interactions. We tested this hypothesis by introducing an epidermal-specific Ikka (also known as Chuk) transgene into IKK-alpha-deficient mice. Mice lacking IKK-alpha in all cell types including bone and cartilage, but not in basal epidermal keratinocytes, exhibit normal epidermal differentiation and skeletal morphology. Thus, epidermal differentiation is required for proper morphogenesis of mesodermally derived skeletal elements. One way by which IKK-alpha controls skeletal and craniofacial morphogenesis is by repressing expression of fibroblast growth factor (FGF) family members, such as FGF8, whose expression is specifically elevated in the limb bud ectoderm of IKK-alpha-deficient mice.
Mitogenic activation of expression of immediate-early genes, such as c-fos, is controlled through signal-induced phosphorylation of constitutively bound transcription factors that is correlated with a nucleosomal response that involves inducible chromatin modifications, such as histone phosphorylation and acetylation. Here we have explored a potential role for the transcription factor NF-kappaB and its associated signaling components in mediating induction of c-fos gene expression downstream of epidermal growth factor (EGF)-dependent signaling. Here we show that EGF treatment of quiescent fibroblast does not induce the classical pathway of NF-kappaB activation through IkappaB kinase (IKK)-directed IkappaBalpha phosphorylation. Interestingly, efficient induction of c-fos transcription requires IKKalpha, one of the subunits of the IkappaB kinase complex. The NF-kappaB subunit, p65/RelA, is found constitutively associated with the c-fos promoter, and knock-out of this transcription factor significantly reduces c-fos gene expression. Importantly, EGF induces the recruitment of IKKalpha to the c-fos promoter to regulate promoter-specific histone H3 Ser(10) phosphorylation in a manner that is independent of p65/RelA. Collectively, our data demonstrate that IKKalpha and p65/RelA contribute significantly to EGF-induced c-fos gene expression in a manner independent of the classical, IkappaBalpha degradation, p65/RelA nuclear accumulation response pathway.
SRCs (steroid receptor coactivators) are required for nuclear receptor-mediated transcription and are also implicated in the transcription initiation by other transcription factors, such as STATs and NFkappaB. Despite phenotypic manifestations in gene knockout mice for SRC-1, GRIP1, and AIB1 of the SRC (Steroid Receptor Coactivator) family indicating their differential roles in animal physiology, there is no clear evidence, at the molecular level, to support a functional specificity for these proteins. We demonstrated in this report that two species of SRC coactivators, either as AIB1:GRIP1 or as AIB1:SRC-1 are recruited, possibly through heterodimerization, on the promoter of genes that contain a classical hormone responsive element (HRE). In contrast, on non-HRE-containing gene promoters, on which steroid receptors bind indirectly, either GRIP1 or SRC-1 is recruited as a monomer, depending on the cellular abundance of the protein. Typically, non-HRE-containing genes are early genes activated by steroid receptors, whereas HRE-containing genes are activated later. Our results also showed that SRC proteins contribute to the temporal regulation of gene transcription. In addition, our experiments revealed a positive correlation between AIB1/c-myc overexpression in ER+ breast carcinoma samples, suggesting a possible mechanism for AIB1 in breast cancer carcinogenesis.
NF-kappaB is a principal transcriptional regulator of diverse cytokine-mediated processes and is tightly controlled by the IkappaB kinase complex (IKK-alpha/beta/gamma). IKK-beta and IKK-gamma are critical for cytokine-induced NF-kappaB function, whereas IKK-alpha is thought to be involved in other regulatory pathways. However, recent data suggest a role for IKK-alpha in NF-kappaB-dependent gene expression in response to cytokine treatment. Here we demonstrate nuclear accumulation of IKK-alpha after cytokine exposure, suggesting a nuclear function for this protein. Consistent with this, chromatin immunoprecipitation (ChIP) assays reveal that IKK-alpha was recruited to the promoter regions of NF-kappaB-regulated genes on stimulation with tumour-necrosis factor-alpha. Notably, NF-kappaB-regulated gene expression is suppressed by the loss of IKK-alpha and this correlates with a complete loss of gene-specific phosphorylation of histone H3 on serine 10, a modification previously associated with positive gene expression. Furthermore, we show that IKK-alpha can directly phosphorylate histone H3 in vitro, suggesting a new substrate for this kinase. We propose that IKK-alpha is an essential regulator of NF-kappaB-dependent gene expression through control of promoter-associated histone phosphorylation after cytokine exposure. These findings provide additional insight into the role of the IKK complex in NF-kappaB-regulated gene expression.
Cytokine-induced activation of the IkappaB kinases (IKK) IKK-alpha and IKK-beta is a key step involved in the activation of the NF-kappaB pathway. Gene-disruption studies of the murine IKK genes have shown that IKK-beta, but not IKK-alpha, is critical for cytokine-induced IkappaB degradation. Nevertheless, mouse embryo fibroblasts deficient in IKK-alpha are defective in the induction of NF-kappaB-dependent transcription. These observations raised the question of whether IKK-alpha might regulate a previously undescribed step to activate the NF-kappaB pathway that is independent of its previously described cytoplasmic role in the phosphorylation of IkappaBalpha. Here we show that IKK-alpha functions in the nucleus to activate the expression of NF-kappaB-responsive genes after stimulation with cytokines. IKK-alpha interacts with CREB-binding protein and in conjunction with Rel A is recruited to NF-kappaB-responsive promoters and mediates the cytokine-induced phosphorylation and subsequent acetylation of specific residues in histone H3. These results define a new nuclear role of IKK-alpha in modifying histone function that is critical for the activation of NF-kappaB-directed gene expression.
The IκB kinase (IKK) complex, which is composed of the two kinases IKKα and IKKβ and the regulatory subunit IKKγ/nuclear factor-κB
(NF-κB) essential modulator (NEMO), is important in the cytokine-induced activation of the NF-κB pathway. In addition to modulation
of IKK activity, the NF-κB pathway is also regulated by other processes, including the nucleocytoplasmic shuttling of various
components of this pathway and the post-translational modification of factors bound to NF-κB-dependent promoters. In this
study, we explored the role of the nucleocytoplasmic shuttling of components of the IKK complex in the regulation of the NF-κB
pathway. IKKγ/NEMO was demonstrated to shuttle between the cytoplasm and the nucleus and to interact with the nuclear coactivator
cAMP-responsive element-binding protein-binding protein (CBP). Using both in vitro and in vivo analysis, we demonstrated that IKKγ/NEMO competed with p65 and IKKα for binding to the N terminus of CBP, inhibiting CBP-dependent
transcriptional activation. These results indicate that, in addition to the key role of IKKγ/NEMO in regulating cytokine-induced
IKK activity, its ability to shuttle between the cytoplasm and the nucleus and to bind to CBP can lead to transcriptional
repression of the NF-κB pathway.
Processing of the nfκb2 gene product p100 to generate p52 is an important step in NF-κB regulation. We show that this step is negatively regulated by a processing-inhibitory domain (PID) within p100 and positively regulated by the NF-κB-inducing kinase (NIK). While the PID suppresses the constitutive processing of p100, NIK induces p100 processing by stimulating site-specific phosphorylation and ubiquitination of this precursor protein. Further, a natural mutation of the gene encoding NIK in alymphoplasia (aly) mice cripples the function of NIK in p100 processing, causing a severe defect in p52 production. These data suggest that NIK is a specific kinase regulating p100 processing and explain why the aly and nfκb2 knockout mice exhibit similar immune deficiencies.
The regulation of the transcription factor NF-κB activity occurs at several levels including controlled cytoplasmic-nuclear shuttling and modulation of its transcriptional activity. A critical component in NF-κB regulation is the IκB kinase (IKK) complex. This review is focused on recent progress as well as unanswered questions regarding the regulation and function of NF-κB and IKK.
The oligomeric IκB kinase (IKK) is composed of three polypeptides: IKKα and IKKβ, the catalytic subunits, and IKKγ, a regulatory
subunit. IKKα and IKKβ are similar in structure and thought to have similar function—phosphorylation of the IκB inhibitors
in response to proinflammatory stimuli. Such phosphorylation leads to degradation of IκB and activation of nuclear factor
κB transcription factors. The physiological function of these protein kinases was explored by analysis of IKKα-deficient mice.
IKKα was not required for activation of IKK and degradation of IκB by proinflammatory stimuli. Instead, loss of IKKα interfered
with multiple morphogenetic events, including limb and skeletal patterning and proliferation and differentiation of epidermal
keratinocytes.
The gene encoding inhibitor of kappa B (IκB) kinase α (IKKα; also called IKK1) was disrupted by gene targeting. IKKα-deficient
mice died perinatally. In IKKα-deficient fetuses, limb outgrowth was severely impaired despite unaffected skeletal development.
The epidermal cells in IKKα-deficient fetuses were highly proliferative with dysregulated epidermal differentiation. In the
basal layer, degradation of IκB and nuclear localization of nuclear factor kappa B (NF-κB) were not observed. Thus, IKKα is
essential for NF-κB activation in the limb and skin during embryogenesis. In contrast, there was no impairment of NF-κB activation
induced by either interleukin-1 or tumor necrosis factor–α in IKKα-deficient embryonic fibroblasts and thymocytes, indicating
that IKKα is not essential for cytokine-induced activation of NF-κB.
The ability of estrogens to stimulate the transcriptional activity of the estrogen receptor can be inhibited by a diverse range of estrogen antagonists. Here we show that the antiestrogen ICI 164,384, N-(n-butyl)-11-[3,17 beta-dihydroxy-estra-1,3,5(10)-trien-7 alpha-yl]N-methylundecanamide, rapidly reduces the levels of receptor protein transiently expressed in cells without affecting receptor mRNA abundance. The reduction in the levels of receptor protein is dose dependent, reversible by estradiol, and mediated by the hormone-binding domain of the receptor. Pulse-chase experiments indicate that the half-life of the receptor is reduced from approximately 5 hr in the presence of estradiol to less than 1 hr by ICI 164,384. A similar reduction in estrogen receptor levels is demonstrated in human breast cancer cells treated with ICI 164,384. We discuss the possibility that the increased turnover of the receptor might be a consequence of impaired receptor dimerization.
Mice lacking cyclin D1 have been generated by gene targeting in embryonic stem cells. Cyclin D1-deficient animals develop to term but show reduced body size, reduced viability, and symptoms of neurological impairment. Their retinas display a striking reduction in cell number due to proliferative failure during embryonic development. In situ hybridization studies of normal mouse embryos revealed an extremely high level of cyclin D1 in the retina, suggesting a special dependence of this tissue on cyclin D1. In adult mutant females, the breast epithelial compartment fails to undergo the massive proliferative changes associated with pregnancy despite normal levels of ovarian steroid hormones. Thus, steroid-induced proliferation of mammary epithelium during pregnancy may be driven through cyclin D1.
Cyclins and proto-oncogenes including c-myc have been implicated in eukaryotic cell cycle control. The role of cyclins in steroidal regulation of cell proliferation is unknown, but a role for c-myc has been suggested. This study investigated the relationship between regulation of T-47D breast cancer cell cycle progression, particularly by steroids and their antagonists, and changes in the levels of expression of these genes. Sequential induction of cyclins D1 (early G1 phase), D3, E, A (late G1-early S phase), and B1 (G2 phase) was observed following insulin stimulation of cell cycle progression in serum-free medium. Transient acceleration of G1-phase cells by progestin was also accompanied by rapid induction of cyclin D1, apparent within 2 h. This early induction of cyclin D1 and the ability of delayed administration of antiprogestin to antagonize progestin-induced increases in both cyclin D1 mRNA and the proportion of cells in S phase support a central role for cyclin D1 in mediating the mitogenic response in T-47D cells. Compatible with this hypothesis, antiestrogen treatment reduced the expression of cyclin D1 approximately 8 h before changes in cell cycle phase distribution accompanying growth inhibition. In the absence of progestin, antiprogestin treatment inhibited T-47D cell cycle progression but in contrast did not decrease cyclin D1 expression. Thus, changes in cyclin D1 gene expression are often, but not invariably, associated with changes in the rate of T-47D breast cancer cell cycle progression. However, both antiestrogen and antiprogestin depleted c-myc mRNA by > 80% within 2 h. These data suggest the involvement of both cyclin D1 and c-myc in the steroidal control of breast cancer cell cycle progression.
Both cyclin D1 and estrogens have an essential role in regulating proliferation of breast epithelial cells. We show here a novel role for cyclin D1 in growth regulation of estrogen-responsive tissues by potentiating transcription of estrogen receptor-regulated genes. Cyclin D1 mediates this activation independent of complex formation to a CDK partner. Cyclin D1 activates estrogen receptor-mediated transcription in the absence of estrogen and enhances transcription in its presence. The activation of estrogen receptor by cyclin D1 is not inhibited by anti-estrogens. A direct physical binding of cyclin D1 to the hormone binding domain of the estrogen receptor results in an increased binding of the receptor to estrogen response element sequences, and upregulates estrogen receptor-mediated transcription. These results highlight a novel role for cyclin D1 as a CDK-independent activator of the estrogen receptor.
The oligomeric IkappaB kinase (IKK) is composed of three polypeptides: IKKalpha and IKKbeta, the catalytic subunits, and IKKgamma, a regulatory subunit. IKKalpha and IKKbeta are similar in structure and thought to have similar function-phosphorylation of the IkappaB inhibitors in response to proinflammatory stimuli. Such phosphorylation leads to degradation of IkappaB and activation of nuclear factor kappaB transcription factors. The physiological function of these protein kinases was explored by analysis of IKKalpha-deficient mice. IKKalpha was not required for activation of IKK and degradation of IkappaB by proinflammatory stimuli. Instead, loss of IKKalpha interfered with multiple morphogenetic events, including limb and skeletal patterning and proliferation and differentiation of epidermal keratinocytes.
The gene encoding inhibitor of kappa B (IkappaB) kinase alpha (IKKalpha; also called IKK1) was disrupted by gene targeting. IKKalpha-deficient mice died perinatally. In IKKalpha-deficient fetuses, limb outgrowth was severely impaired despite unaffected skeletal development. The epidermal cells in IKKalpha-deficient fetuses were highly proliferative with dysregulated epidermal differentiation. In the basal layer, degradation of IkappaB and nuclear localization of nuclear factor kappa B (NF-kappaB) were not observed. Thus, IKKalpha is essential for NF-kappaB activation in the limb and skin during embryogenesis. In contrast, there was no impairment of NF-kappaB activation induced by either interleukin-1 or tumor necrosis factor-alpha in IKKalpha-deficient embryonic fibroblasts and thymocytes, indicating that IKKalpha is not essential for cytokine-induced activation of NF-kappaB.
Many cofactors bind the hormone-activated estrogen receptor (ER), yet the specific regulators of endogenous ER-mediated gene transcription are unknown. Using chromatin immunoprecipitation (ChIP), we find that ER and a number of coactivators rapidly associate with estrogen responsive promoters following estrogen treatment in a cyclic fashion that is not predicted by current models of hormone activation. Cycles of ER complex assembly are followed by transcription. In contrast, the anti-estrogen tamoxifen (TAM) recruits corepressors but not coactivators. Using a genetic approach, we show that recruitment of the p160 class of coactivators is sufficient for gene activation and for the growth stimulatory actions of estrogen in breast cancer supporting a model in which ER cofactors play unique roles in estrogen signaling.
There was a time when the classification of sex hormones was simple. Androgens were male and estrogens female. What remains true today is that in young adults androgen levels are higher in males and estrogen levels higher in females. More recently we have learned that estrogens are necessary in males for regulation of male sexual behavior, maintenance of the skeleton and the cardiovascular system, and for normal function of the testis and prostate. The importance of androgen in females was never in doubt, it is after all the precursor of estrogen as the substrate for aromatase, the enzyme that produces estrogen. In addition, the tissue distribution of androgen receptors suggests that androgens themselves are important in the ovary, uterus, breast, and brain. New information promises to clarify some of the complex issues of the physiological roles of estrogen and the contribution of estrogen to the development of neoplastic diseases in humans. The discovery of the second estrogen receptor, the creation of mutant mice defective in both estrogen receptors and in the aromatase gene, the solution of the structures of the ligand-binding domains of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta), the finding of novel routes through which estrogen receptors can modulate transcription, and the identification of a man with a bi-allelic disruptive mutation of the ERalpha gene are but some of the milestones. This review focuses on the mechanistic aspects of signal transduction mediated by ERs and on the physiological consequences of deficiency of estrogen or estrogen receptor in the available mouse models.
Processing of the nf(kappa)b2 gene product p100 to generate p52 is an important step in NF-kappaB regulation. We show that this step is negatively regulated by a processing-inhibitory domain (PID) within p100 and positively regulated by the NF-kappaB-inducing kinase (NIK). While the PID suppresses the constitutive processing of p100, NIK induces p100 processing by stimulating site-specific phosphorylation and ubiquitination of this precursor protein. Further, a natural mutation of the gene encoding NIK in alymphoplasia (aly) mice cripples the function of NIK in p100 processing, causing a severe defect in p52 production. These data suggest that NIK is a specific kinase regulating p100 processing and explain why the aly and nf(kappa)b2 knockout mice exhibit similar immune deficiencies.
In mammals, the canonical nuclear factor κB (NF-κB) signaling pathway activated in response to infections is based on degradation
of IκB inhibitors. This pathway depends on the IκB kinase (IKK), which contains two catalytic subunits, IKKα and IKKβ. IKKβ
is essential for inducible IκB phosphorylation and degradation, whereas IKKα is not. Here we show that IKKα is required for
B cell maturation, formation of secondary lymphoid organs, increased expression of certain NF-κB target genes, and processing
of the NF-κB2 (p100) precursor. IKKα preferentially phosphorylates NF-κB2, and this activity requires its phosphorylation
by upstream kinases, one of which may be NF-κB–inducing kinase (NIK). IKKα is therefore a pivotal component of a second NF-κB
activation pathway based on regulated NF-κB2 processing rather than IκB degradation.
To identify functions of the IKKalpha subunit of IkappaB kinase that require catalytic activity, we generated an Ikkalpha(AA) knockin allele containing alanines instead of serines in the activation loop. Ikkalpha(AA/AA) mice are healthy and fertile, but females display a severe lactation defect due to impaired proliferation of mammary epithelial cells. IKKalpha activity is required for NF-kappaB activation in mammary epithelial cells during pregnancy and in response to RANK ligand but not TNFalpha. IKKalpha and NF-kappaB activation are also required for optimal cyclin D1 induction. Defective RANK signaling or cyclin D1 expression results in the same phenotypic effect as the Ikkalpha(AA) mutation, which is completely suppressed by a mammary specific cyclin D1 transgene. Thus, IKKalpha is a critical intermediate in a pathway that controls mammary epithelial proliferation in response to RANK signaling via cyclin D1.
The nuclear receptor (NR) superfamily of transcription factors regulates gene expression in response to endocrine signaling, and recruitment of coregulators affords these receptors considerable functional flexibility. We will place historical aspects of NR research in context with current opinions on their mechanism of signal transduction, and we will speculate upon future trends in the field.
Selective estrogen receptor modulators (SERMs) mimic estrogen action in certain tissues while opposing it in others. The therapeutic
effectiveness of SERMs such as tamoxifen and raloxifene in breast cancer depends on their antiestrogenic activity. In the
uterus, however, tamoxifen is estrogenic. Here, we show that both tamoxifen and raloxifene induce the recruitment of corepressors
to target gene promoters in mammary cells. In endometrial cells, tamoxifen, but not raloxifene, acts like estrogen by stimulating
the recruitment of coactivators to a subset of genes. The estrogen-like activity of tamoxifen in the uterus requires a high
level of steroid receptor coactivator 1 (SRC-1) expression. Thus cell type– and promoter-specific differences in coregulator
recruitment determine the cellular response to SERMs.
The regulation of the transcription factor NF-kappaB activity occurs at several levels including controlled cytoplasmic-nuclear shuttling and modulation of its transcriptional activity. A critical component in NF-kappaB regulation is the IkappaB kinase (IKK) complex. This review is focused on recent progress as well as unanswered questions regarding the regulation and function of NF-kappaB and IKK.
Estrogen receptor alpha (ERalpha) is phosphorylated on multiple amino acid residues. For example, in response to estradiol binding, human ERalpha is predominately phosphorylated on Ser-118 and to a lesser extent on Ser-104 and Ser-106. In response to activation of the mitogen-activated protein kinase pathway, phosphorylation occurs on Ser-118 and Ser-167. These serine residues are all located within the activation function 1 region of the N-terminal domain of ERalpha. In contrast, activation of protein kinase A increases the phosphorylation of Ser-236, which is located in the DNA-binding domain. The in vivo phosphorylation status of Tyr-537, located in the ligand-binding domain, remains controversial. In this review, I present evidence that these phosphorylations occur, and identify the kinases thought to be responsible. Additionally, the functional importance of ERalpha phosphorylation is discussed.
Cytokine treatment stimulates the IkappaB kinases, IKKalpha and IKKbeta, which phosphorylate the IkappaB proteins, leading to their degradation and activation of NF-kappaB regulated genes. A clear definition of the specific roles of IKKalpha and IKKbeta in activating the NF-kappaB pathway and the upstream kinases that regulate IKK activity remain to be elucidated. Here, we utilized small interfering RNAs (siRNAs) directed against IKKalpha, IKKbeta and the upstream regulatory kinase TAK1 in order to better define their roles in cytokine-induced activation of the NF-kappaB pathway. In contrast to previous results with mouse embryo fibroblasts lacking either IKKalpha or IKKbeta, which indicated that only IKKbeta is involved in cytokine-induced NF-kappaB activation, we found that both IKKalpha and IKKbeta were important in activating the NF-kappaB pathway. Furthermore, we found that the MAP3K TAK1, which has been implicated in IL-1-induced activation of the NF-kappaB pathway, was also critical for TNFalpha-induced activation of the NF-kappaB pathway. TNFalpha activation of the NF-kappaB pathway is associated with the inducible binding of TAK1 to TRAF2 and both IKKalpha and IKKbeta. This analysis further defines the distinct in vivo roles of IKKalpha, IKKbeta and TAK1 in cytokine-induced activation of the NF-kappaB pathway.
Transcriptional activation of a gene involves an orchestrated recruitment of components of the basal transcription machinery and intermediate factors, concomitant with an alteration in local chromatin structure generated by posttranslational modifications of histone tails and nucleosome remodeling. We provide here a comprehensive picture of events resulting in transcriptional activation of a gene, through evaluating the estrogen receptor-alpha (NR3A1) target pS2 gene promoter in MCF-7 cells. This description integrates chromatin remodeling with a kinetic evaluation of cyclical networks of association of 46 transcription factors with the promoter, as determined by chromatin immunoprecipitation assays. We define the concept of a "transcriptional clock" that directs and achieves the sequential and combinatorial assembly of a transcriptionally productive complex on a promoter. Furthermore, the unanticipated findings of key roles for histone deacetylases and nucleosome-remodeling complexes in limiting transcription implies that transcriptional activation is a cyclical process that requires both activating and repressive epigenetic processes.
The nuclear factor (NF)-kappaB pathway is important for the expression of a wide variety of genes that are involved in the control of the host immune and inflammatory response, and in the regulation of cellular proliferation and survival. The constitutive activation of this pathway is associated with inflammatory and autoimmune diseases, such as asthma, rheumatoid arthritis and inflammatory bowel disease, in addition to atherosclerosis, Alzheimer's disease, cancer and diabetes. One of the key steps in activating the NF-kappaB pathway is the stimulation of the IkappaB (inhibitor of kappaB) kinases. Recent data indicate that these kinases activate the NF-kappaB pathway through distinct steps that are operative in both the cytoplasm and the nucleus. A better understanding of the mechanisms that activate this pathway provides the potential for defining new therapeutic targets that might prevent the aberrant activation of NF-kappaB in a variety of human diseases.
Although several lines of evidence have indicated that the activity of SRC-3/AIB1/ACTR/pCIP/RAC3/TRAM1 could be regulated by phosphorylation, an important question remained as to how different signaling pathways can act through limiting concentrations of the same SRC-3 molecule to exert different physiological functions. Herein, we report the successful identification of six functional in vivo SRC-3 phosphorylation sites. Interestingly, all phosphorylation sites are required for coactivation of estrogen and androgen receptors, but not all sites are required for coactivation of NF-kappaB. Different combinations of site-specific phosphorylations of SRC-3 are required for induction of IL-6 gene expression by TNF-alpha as compared to oncogenic transformation of MEFs. Mechanisms of pathway selectivity involve protein-protein interactions of differentially phosphorylated SRC-3 with downstream transcriptional activators and coactivators. Our results uncovered an additional level of transcriptional regulation whereby specific modulations of SRC-3 phosphorylation allow this coactivator to function as a regulatable integrator for diverse signaling pathways in cells.
Understanding how signaling cascades stimulate chromatin-remodeling events through derepression is one of the foremost questions in the transcription field. Here, we demonstrate that NF-kappaB transcription requires IKKalpha to phosphorylate SMRT on chromatin, stimulating the exchange of corepressor for coactivator complexes. IKKalpha-induced phosphorylation coincides with a loss of chromatin-associated SMRT and HDAC3 and with nuclear export of the SMRT corepressor, events required for expression of the NF-kappaB-regulated cIAP-2 and IL-8 genes. Although SMRT derepression corresponds with the recruitment of TBL1/TBLR1, this complex alone is insufficient to relieve repression. Using a nonphosphorylatable SMRT protein, we demonstrate that IKKalpha-induced phosphorylation is required to recruit 14-3-3epsilon and Ubc5 for SMRT derepression. Failure of IKKalpha to stimulate the removal of SMRT from chromatin inhibits the recruitment of NF-kappaB to promoters, blocking transcription and sensitizing cells to apoptosis. Our work provides evidence that IKKalpha orchestrates SMRT derepression, a prerequisite for NF-kappaB transcription and survival.
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