Activator Protein 1 Dimer Research Articles

Liver cancer development driven by the AP-1/c-Jun~Fra-2 dimer through c-Myc

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. HCC incidence is on the rise, while treatment options remain limited. Thus, a better understanding of the molecular pathways involved in HCC development has become a priority to guide future therapies. While previous studies implicated the Activator Protein-1 (AP-1) (Fos/Jun) transcription factor family members c-Fos and c-Jun in HCC formation, the contribution of Fos-related antigens (Fra-) 1 and 2 is unknown. Here, we show that hepatocyte-restricted expression of a single chain c-Jun~Fra-2 protein, which functionally mimics the c-Jun/Fra-2 AP-1 dimer, results in spontaneous HCC formation in c-Jun~Fra-2hep mice. Several hallmarks of human HCC, such as cell cycle dysregulation and the expression of HCC markers are observed in liver tumors arising in c-Jun~Fra-2hep mice. Tumorigenesis occurs in the context of mild inflammation, low-grade fibrosis, and Pparγ-driven dyslipidemia. Subsequent analyses revealed increased expression of c-Myc, evidently under direct regulation by AP-1 through a conserved distal 3' enhancer. Importantly, c-Jun~Fra-2-induced tumors revert upon switching off transgene expression, suggesting oncogene addiction to the c-Jun~Fra-2 transgene. Tumors escaping reversion maintained c-Myc and c-Myc target gene expression, likely due to increased c-Fos. Interfering with c-Myc in established tumors using the Bromodomain and Extra-Terminal motif inhibitor JQ-1 diminished liver tumor growth in c-Jun~Fra-2 mutant mice. Thus, our data establish c-Jun~Fra-2hep mice as a model to study liver tumorigenesis and identify the c-Jun/Fra-2-Myc interaction as a potential target to improve HCC patient stratification and/or therapy.

Methyl-dependent and spatial-specific DNA recognition by the orthologous transcription factors human AP-1 and Epstein-Barr virus Zta.

Activator protein 1 (AP-1) is a transcription factor that recognizes two versions of a 7-base pair response element, either 5΄-TGAGTCA-3΄ or 5΄-MGAGTCA-3΄ (where M = 5-methylcytosine). These two elements share the feature that 5-methylcytosine and thymine both have a methyl group in the same position, 5-carbon of the pyrimidine, so each of them has two methyl groups at nucleotide positions 1 and 5 from the 5΄ end, resulting in four methyl groups symmetrically positioned in duplex DNA. Epstein-Barr Virus Zta is a key transcriptional regulator of the viral lytic cycle that is homologous to AP-1. Zta recognizes several methylated Zta-response elements, including meZRE1 (5΄-TGAGMCA-3΄) and meZRE2 (5΄-TGAGMGA-3΄), where a methylated cytosine occupies one of the inner thymine residues corresponding to the AP-1 element, resulting in the four spatially equivalent methyl groups. Here, we study how AP-1 and Zta recognize these methyl groups within their cognate response elements. These methyl groups are in van der Waals contact with a conserved di-alanine in AP-1 dimer (Ala265 and Ala266 in Jun), or with the corresponding Zta residues Ala185 and Ser186 (via its side chain carbon Cβ atom). Furthermore, the two ZRE elements differ at base pair 6 (C:G versus G:C), forming a pseudo-symmetric sequence (meZRE1) or an asymmetric sequence (meZRE2). In vitro DNA binding assays suggest that Zta has high affinity for all four sequences examined, whereas AP-1 has considerably reduced affinity for the asymmetric sequence (meZRE2). We ascribe this difference to Zta Ser186 (a unique residue for Zta) whose side chain hydroxyl oxygen atom interacts with the two half sites differently, whereas the corresponding Ala266 of AP-1 Jun protein lacks such flexibility. Our analyses demonstrate a novel mechanism of 5mC/T recognition in a methylation-dependent, spatial and sequence-specific approach by basic leucine-zipper transcriptional factors.

Identification of FRA-1 as a novel player in pancreatic cancer in cooperation with a MUC1: ERK signaling axis.

The MUC1 glycoprotein is overexpressed and aberrantly glycosylated in >90% of pancreatic ductal adenocarcinoma cases and impacts tumor progression by initiating downstream signaling through phosphorylation of its cytoplasmic tail. Previous studies have demonstrated that MUC1 alters expression of known targets of activator protein 1 (AP-1); however, no studies have evaluated the precise impact of MUC1 signaling on the activity and formation of AP-1. Given the known role of these proteins in modulating migration, invasion, and tumor progression, we explored the effects of MUC1 on AP-1 dimer formation and function. We determined that MUC1 increased the protein levels of c-Jun, the major component of AP-1, and promoted dimerization of c-Jun with the Fos-protein FRA-1. We demonstrate that FRA-1 acts as a potent mediator of migration and invasion in a manner that is modulated by signals through MUC1, which acts as a dominant regulator of specific AP-1 and FRA-1 target genes. Our results provide the first in vivo evidence of a FRA-1 mediated expression profile that impacts pancreatic tumor growth properties. In summary, we show that MUC1 enhancement of ERK activation influences FRA-1 activity to modulate tumor migration, invasion and metastasis in a subset of pancreatic cancer cases.

HDAC inhibitors suppress c-Jun/Fra-1-mediated proliferation through transcriptionally downregulating MKK7 and Raf1 in neuroblastoma cells.

Activator protein 1 (AP-1) is a transcriptional factor composed of the dimeric members of bZIP proteins, which are frequently deregulated in human cancer cells. In this study, we aimed to identify an oncogenic AP-1 dimer critical for the proliferation of neuroblastoma cells and to investigate whether histone deacetylase inhibitors (HDACIs), a new generation of anticancer agents, could target the AP-1 dimer. We report here that HDACIs including trichostatin A, suberoylanilidehydroxamic acid, valproic acid and M344 can transcriptionally suppress both c-Jun and Fra-1, preceding their inhibition of cell growth. c-Jun preferentially interacting with Fra-1 as a heterodimer is responsible for AP-1 activity and critical for cell growth. Mechanistically, HDACIs suppress Fra-1 expression through transcriptionally downregulating Raf1 and subsequently decreasing MEK1/2-ERK1/2 activity. Unexpectedly, HDACI treatment caused MKK7 downregulation at both the protein and mRNA levels. Deletion analysis of the 5′-flanking sequence of the MKK7 gene revealed that a major element responsible for the downregulation by HDACI is located at −149 to −3 relative to the transcriptional start site. Knockdown of MKK7 but not MKK4 remarkably decreased JNK/c-Jun activity and proliferation, whereas ectopic MKK7-JNK1 reversed HDACI-induced c-Jun suppression. Furthermore, suppression of both MKK-7/c-Jun and Raf-1/Fra-1 activities was involved in the tumor growth inhibitory effects induced by SAHA in SH-SY5Y xenograft mice. Collectively, these findings demonstrated that c-Jun/Fra-1 dimer is critical for neuroblastoma cell growth and that HDACIs act as effective suppressors of the two oncogenes through transcriptionally downregulating MKK7 and Raf1.

Counter-regulation of the AP-1 monomers pATF2 and Fos: Molecular readjustment of brainstem neurons in hearing and deaf adult rats after electrical intracochlear stimulation

Expression of the immediate-early gene fos (also known as c-fos) and phosphorylation of the product of the early response gene atf2 (pATF2) in the adult auditory brainstem can be modulated by electrical intracochlear stimulation. The Fos and pATF2 proteins are competitive monomers of the heterodimeric activator protein-1 (AP-1) transcription factor that triggers the expression of genes related to neural plasticity. Our previous findings showed that the stimulation-induced spatio-temporal pattern of Fos expression in the adult auditory system depends on hearing experience. In this study, we aimed to identify a possible correlation of pATF2 and Fos expression. Adult normal hearing and neonatally deafened rats were unilaterally stimulated with a cochlear implant (CI) for 45min, 73min, or 2h. The numbers of Fos- and pATF2-positive neurons in the anteroventral cochlear nucleus (AVCN), the lateral superior olive (LSO), and the central inferior colliculus (CIC) were evaluated. Following stimulation, an increased Fos expression was demonstrated in all these regions in hearing and deaf rats. However, in neonatally deafened rats, significantly more Fos-positive neurons emerged that did not obey a tonotopic order. Independent of hearing experience, Fos expression correlated with a locally matching decrease of pATF2 expression in AVCN and LSO, but not in CIC. We suggest that these changes in gene expression result in a shift of AP-1 dimer composition from ATF2:Jun to Fos:Jun. This change in AP-1 constellation is expected to invoke different transcriptional cascades leading to distinct modes of tissue reorganization and plasticity responses in the mature central auditory system under stimulation.

Abstract 508: Identification of FRA-1 as a potential driver of pro-invasive properties in pancreatic cancer in conjunction with MUC1

Abstract The transmembrane mucin, MUC1, contributes to the progression of pancreatic cancer, in part by conducting signals from the cell surface that regulate expression of molecules associated with aggressive tumor behavior. We investigated transcriptional co-regulatory effects mediated by interactions of the MUC1 cytoplasmic tail with activator protein 1 (AP-1). Previous results suggest that MUC1 regulates the capacity of AP-1 to bind specific promoter elements and regulate genes that contribute to tumor progression. The DNA binding activity of AP-1 is controlled in several ways including composition of the protein dimer. The precise mechanism by which MUC1 alters transcriptional complexes remains unknown. We sought to determine how MUC1 influences the formation of AP-1 complexes and potential downstream effects. We found MUC1 increased c-Jun protein levels, the major component of AP-1, in a manner largely independent of transcriptional upregulation (mRNA upregulation was observed in only one cell line of several analyzed). Formation of the AP-1 dimer was assessed by quantitative and semi-quantitative assays. MUC1 promoted the association of c-Jun with the Fos protein, FRA-1. This association was promoted by enhanced ERK2 signaling in MUC1 expressing cell lines. In breast cancer, FRA-1 drives an invasive and metastatic phenotype. Overexpression of FRA-1 in pancreatic cancer cell lines promoted migratory and invasive behaviors in vitro. Blocking FRA-1 activity by shRNA knockdown or ERK inhibition decreased migration and invasion. Analysis of gene expression in pancreatic tumor samples and normal, uninvolved tissue [mined from the Gene Expression Omnibus (GEO)] revealed that tumor samples had significantly increased levels of FRA-1 mRNA. A subset of these samples exhibited gene expression patterns consistent with a FRA-1:EMT phenotype previously observed in colorectal cancer cells. We evaluated expression of FRA-1 protein in primary pancreatic tumors, liver metastases, and uninvolved non-malignant pancreas. Robust nuclear localization of FRA-1 was observed in primary and metastatic tumor cells whereas uninvolved pancreas showed lower levels of diffuse cytoplasmic staining. Based on these results, we hypothesize that FRA-1 plays an important role in a subset of pancreatic cancers by altering the binding profile of AP-1. Due to a dependence on ERK activity for FRA-1 function, we propose that cancers with activating mutations in the RAS-RAF-MEK-ERK cascade are the most likely candidates for utilization of FRA-1. Citation Format: Ryan L. Hanson, Michael A. Hollingsworth. Identification of FRA-1 as a potential driver of pro-invasive properties in pancreatic cancer in conjunction with MUC1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 508. doi:10.1158/1538-7445.AM2015-508

Inhibition of Activator Protein-1 by Sulforaphane Involves Interaction with Cysteine in the cFos DNA-Binding Domain: Implications for Chemoprevention of UVB-Induced Skin Cancer

Sulforaphane is an isothiocyanate derived from cruciferous vegetables that has been linked to decreased risk of certain cancers. Although the role of sulforaphane in the induction of the transcription factor Nrf2 has been studied extensively, there is also evidence that inhibition of the transcription factor activator protein-1 (AP-1) may contribute to the chemopreventive properties of this compound. In this study, we show for the first time that sulforaphane is effective at reducing the multiplicity and tumor burden of UVB-induced squamous cell carcinoma in a mouse model using cotreatment with the compound and the carcinogen. We also show that sulforaphane pretreatment is able to reduce the activity of AP-1 luciferase in the skin of transgenic mice after UVB. Chromatin immunoprecipitation analysis verified that a main constituent of the AP-1 dimer, cFos, is inhibited from binding to the AP-1 DNA binding site by sulforaphane. Electrophoretic mobility shift assay analysis of nuclear proteins also shows that sulforaphane and diamide, both known to react with cysteine amino acids, are effective at inhibiting AP-1 from binding to its response element. Using truncated recombinant cFos and cJun, we show that mutation of critical cysteines in the DNA-binding domain of these proteins (Cys(154) in cFos and Cys(272) in cJun) results in loss of sensitivity to both sulforaphane and diamide in electrophoretic mobility shift assay analysis. Together, these data indicate that inhibition of AP-1 activity may be an important molecular mechanism in chemoprevention of squamous cell carcinoma by sulforaphane.

Dimer Composition and Promoter Context Contribute to Functional Cooperation between AP-1 and NFAT

The transcription factors activator protein 1 (AP-1) and nuclear factor of activated T-cells (NFAT) cooperate to induce the expression of cytokines during the immune response. While much is known about the signaling pathways and physical interactions between NFAT and AP-1 dimers following lymphocyte activation, few studies have addressed the role of AP-1 composition in modulating NFAT:AP-1-dependent transcription. We examined the function of specific AP-1 complexes using “tethered” AP-1 dimers with defined composition. We found that NFAT can functionally cooperate with all AP-1 dimers tested. Noteworthy, Jun∼Jun-containing dimers, which are relatively inactive when tested on an AP-1-dependent promoter, are effective co-activators of an NFAT:AP-1-dependent promoter. Interestingly, specific AP-1 dimer combinations behave differently when tested on interleukin 2 ( IL2) and interleukin 4 ( IL4) gene regulatory regions. Moreover, the requirement for NFAT to activate each of the promoters is different. Our results suggest that higher NFAT levels are necessary to activate the IL4 promoter. Hence changes in AP-1 composition and the level of participating NFAT proteins can differentially influence cytokine gene expression, resulting in biological consequences for the modulation and dynamics of the immune response.

MEKK1 regulates the AP-1 dimer repertoire via control of JunB transcription and Fra-2 protein stability

Activator protein 1 (AP-1) transcription factor dimers are composed of Jun, Fos, and ATF member proteins, but the mechanisms that determine AP-1 composition are not clearly defined and the function of specific dimers is not well understood. MEKK1 is a mitogen-activated protein kinase (MAPK) kinase kinase and an ubiquitin ligase that regulates both the extracellular signal-regulated kinase 1/2 and the c-Jun amino-terminal kinase. Herein, we demonstrate that MEKK1 regulates the AP-1 protein repertoire. Both FGF-2 and phorbol ester-inducible urokinase-type plasminogen activator (uPA) expression requires AP-1 binding to an enhancer element in the uPA promoter, and we have previously shown that FGF-2 or PMA induction of uPA expression is strongly dependent on MEKK1. JunB mRNA is significantly increased in MEKK1-/- cells, demonstrating that MEKK1 suppresses JunB mRNA expression. Upregulation of JunB expression in MEKK1-/- cells forms an inhibitory AP-1 complex that binds to the uPA promoter and inhibits uPA transcription. MEKK1 also regulates Fra-2 protein stability by inducing Fra-2 ubiquitination and degradation. MEKK1 regulates AP-1-dependent gene expression by regulating the expression, activity and degradation of component members of the AP-1 complex. Controlling the repertoire of a transcription factor complex is a newly defined function for an MAPK kinase kinase.