Fra-1 Protein Research Articles

Expression of Fra-1 in laryngeal squamous cell carcinoma

Objective:To investigate expressions of Fra-1 in laryngeal squamous cell carcinoma. Method:Using immunohistochemical method and RT-PCR detecting 47 cases of laryngeal squamous carcinoma tissues and 21 cases of para-carcinoma tissues with the level of protein and mRNA, we analyzed the relationship of the genes expression and clinical parameters. Result:The level of protein positive expression of Fra-1 genes in laryngeal squamous cancer tissue were higher than adiacent tissues (P<0.05). The expression of Fra-1 protein in laryngeal squamous cancer tissue were significant correlation with the clinical stages, lymph metastasis, smoking. They had no significant correlation with the pathological grade, age, anatomic zonation. The mRNA level expression of Fra-1 genes in laryngeal squamous cancer tissue were higher than adjacent tissues (P<0.01). The expression of Fra-1 gene in the mRNA level was correlated with pathologic stage, clinical stage, lymph metastasis, and smoking, but wasn't correlated with age and anatomic zonation. Conclusion:In laryngeal squamous cell carcinoma, the expression of Fra-1 gene are overexpressed. Fra-1 may play a role in the occurrence and development of the laryngeal cancer.

Expression of Fra-1 in human hepatocellular carcinoma and its prognostic significance.

This study aimed to explore the clinical significance and prognostic value of Fra-1 in hepatocellular carcinoma patients after curative resection. Fra-1 expression was investigated using a combination of techniques: immunohistochemistry for 66 samples of hepatocellular carcinoma and quantitative real-time polymerase chain reaction and western blotting assays for 19 matched hepatocellular carcinoma specimens. Fra-1 was present in 38 of 66 (57.6%) tumor tissues, with intense staining in the nuclei. There was also positive staining in 14 of 66 (21.2%) adjacent peritumoral tissues, with weak staining in the cytoplasm. Quantitative real-time polymerase chain reaction and western blotting assays confirmed higher expression of Fra-1 messenger RNA and Fra-1 protein in tumor tissues than adjacent non-tumor tissues for 19 hepatocellular carcinoma samples (p < 0.001). Positive expression of Fra-1 was significantly related to vascular invasion and serum alpha-fetoprotein. Kaplan-Meier survival analysis found that overexpressed Fra-1 was correlated with poor overall survival and disease-free survival. Multivariate analysis identified Fra-1 as an independent prognostic factor. Fra-1 may be involved in the progress of hepatocellular carcinoma and could be a promising molecular candidate in the diagnosis and treatment of hepatocellular carcinoma.

Differential expression of AP-1 transcription factors in human prostate LNCaP and PC-3 cells: role of Fra-1 in transition to CRPC status.

Androgen receptor (AR) signaling axis plays a vital role in the development of prostate and critical in the progression of prostate cancer. Androgen withdrawal initially regresses tumors but eventually develops into aggressive castration-resistant prostate cancer (CRPC). Activator Protein-1 (AP-1) transcription factors are most likely to be associated with malignant transformation in prostate cancer. Hence, to determine the implication of AR and AP-1 in promoting the transition of prostate cancer to the androgen-independent state, we used AR-positive LNCaP and AR-negative PC-3 cells as an in vitro model system. The effect of dihydrotestosterone or anti-androgen bicalutamide on the cell proliferation and viability was assessed by MTT assay. Expression studies on AR, marker genes-PSA, TMPRSS2, and different AP-1 factors were analyzed by semi-quantitative RT-PCR and expressions of AR and Fra-1 proteins were analyzed by Western blotting. Dihydrotestosterone induced the cell proliferation in LNCaP with no effect on PC-3 cells. Bicalutamide decreased the viability of both LNCaP and PC-3 cells. Dihydrotestosterone induced the expression of AR, PSA, c-Jun, and Fra-1 in LNCaP cells, and it was c-Jun and c-Fos in case of PC-3 cells, while bicalutamide decreased their expression. In addition, constitutive activation and non-regulation of Fra-1 by bicalutamide in PC-3 cells suggested that Fra-1, probably a key component, involved in transition of aggressive androgen-independent PC-3 cells with poor prognosis.

Critical Contribution of Nuclear Factor Erythroid 2-related Factor 2 (NRF2) to Electrophile-induced Interleukin-11 Production

Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that plays a crucial role in protection of cells from electrophile-induced toxicity through up-regulating phase II detoxifying enzymes and phase III transporters. We previously reported that oxidative stress induces up-regulation of interleukin-11 (IL-11), a member of the IL-6 family that ameliorates acetaminophen-induced liver toxicity. However, a role for IL-11 in protection of cells from electrophile-induced toxicity remains unclear. Here we show that an environmental electrophile, 1,2-naphthoquinone (1,2-NQ), but not 15d-prostaglandin J2 (PGJ2) or tert-butylhydroxyquinone (tBHQ), induced IL-11 production. Consistent with a crucial role for prolonged ERK activation in H2O2-induced IL-11 production, 1,2-NQ, but not 15d-PGJ2 or tBHQ, elicited prolonged ERK activation. Conversely, inhibition of the ERK pathway by a MEK inhibitor completely blocked 1,2-NQ-induced IL-11 production at both protein and mRNA levels, further substantiating an intimate cross-talk between ERK activation and 1,2-NQ-induced IL-11 production. Promoter analysis of the Il11 gene revealed that two AP-1 sites were essential for 1,2-NQ-induced promoter activities. Among various members of the AP-1 family, Fra-1 was up-regulated by 1,2-NQ, and its up-regulation was blocked by a MEK inhibitor. Although NRF2 was not required for H2O2-induced IL11 up-regulation, NRF2 was essential for 1,2-NQ-induced IL11 up-regulation by increasing Fra-1 proteins possibly through promoting mRNA translation of FOSL1 Finally, intraperitoneal administration of 1,2-NQ induced body weight loss in wild-type mice, which was further exacerbated in Il11ra1-/- mice compared with Il11ra1+/- mice. Together, both Fra-1 and NRF2 play crucial roles in IL-11 production that protects cells from 1,2-NQ intestinal toxicity.

PKCθ-induced phosphorylations control the ability of Fra-1 to stimulate gene expression and cancer cell migration

The AP-1 transcription factor Fra-1 is aberrantly expressed in a large number of cancers and plays crucial roles in cancer development and progression by stimulating the expression of genes involved in these processes. However, the control of Fra-1 transactivation ability is still unclear and here we hypothesized that PKCθ-induced phosphorylation could be necessary to obtain a fully active Fra-1 protein. Using MCF7 stable cells overexpressing equivalent levels of unphosphorylated Fra-1 or PKCθ-phosphorylated Fra-1, we showed that PKCθ-induced phosphorylation of Fra-1 was crucial for the stimulation of MMP1 and IL6 expression. Consistently, we found a significant positive correlation between PRKCQ (coding for PKCθ) and MMP1 mRNA expression levels in human breast cancer samples. PKCθ-induced phosphorylations, in part at T217 and T227 residues, strongly and specifically increased Fra-1 transcriptional activity through the stimulation of Fra-1 transactivation domain, without affecting JUN factors. More importantly, these phosphorylations were required for Fra-1-induced migration of breast cancer cells and phosphorylated Fra-1 expression was enriched at the invasion front of human breast tumors. Taken together, our findings indicate that PKCθ-induced phosphorylation could be important for the function of Fra-1 in cancer progression.

3P - Dissecting the roles of Fra proteins in lung adenocarcinoma

3P - Dissecting the roles of Fra proteins in lung adenocarcinoma

Fluoride-induced c-Fos expression in MC3T3-E1 osteoblastic cells

Excessive systemic exposure to fluoride leads to disturbances of bone homeostasis. c-Fos is known to be essential in bone development by affecting osteoblast and osteoclast differentiation. In this study, we examined the effects of fluoride exposure on c-Fos expression and its regulatory signaling pathways in MC3T3-E1 mouse osteoblast cell line. c-fos mRNA level, c-Fos protein level and c-Fos DNA-binding activity were markedly increased, with a peak at 2 or 4 h, in MC3T3-E1 cells exposed to sodium fluoride (NaF). Fra-1 protein, another member of Fos family, was also elevated, whereas FosB and Fra-2 proteins remained unchanged. NaF further induced phosphorylation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase 1/2 (ERK1/2), ERK5, c-Jun NH2-terminal kinase and p38. NaF-induced expression of c-Fos protein was markedly suppressed with U0126, the inhibitor of both activated and non-activated forms of MAPK/ERK kinase 1/2 (MEK1/2) and BIX02189, the MEK5 inhibitor, but partially with SP600125, the JNK inhibitor and SB203580, the p38 inhibitor. Therefore, ERK1/2 and ERK5 signal transduction pathways are important for accumulating c-Fos. siRNA targeting against the mouse c-fos gene further enhanced NaF-induced up-regulation of osteoprotegerin (OPG), an inhibitor of osteoclastogenesis, suggesting that c-Fos might negatively regulate OPG expression induced by fluoride in osteoblastic cells.

Naringenin targets ERK2 and suppresses UVB-induced photoaging.

A number of natural phytochemicals have anti‐photoaging properties that appear to be mediated through the inhibition of matrix metalloproteinase‐1 (MMP‐1) expression, but their direct target molecule(s) and mechanism(s) remain unclear. We investigated the effect of naringenin, a major flavonoid found in citrus, on UVB‐induced MMP‐1 expression and identified its direct target. The HaCaT human skin keratinocyte cell line and 3‐dimensional (3‐D) human skin equivalent cultures were treated or not treated with naringenin for 1 hr before exposure to UVB. The mechanism and target(s) of naringenin were analysed by kinase assay and multiplex molecular assays. Dorsal skins of hairless mice were exposed to UVB 3 times per week, with a dose of irradiation that was increased weekly by 1 minimal erythema dose (MED; 45 mJ/cm2) to 4 MED over 15 weeks. Wrinkle formation, water loss and water content were then assessed. Naringenin suppressed UVB‐induced MMP‐1 expression and AP‐1 activity, and strongly suppressed UVB‐induced phosphorylation of Fos‐related antigen (FRA)‐1 at Ser265. Importantly, UVB irradiation‐induced FRA1 protein stability was reduced by treatment with naringenin, as well as with a mitogen‐activated protein kinase (MEK) inhibitor. Naringenin significantly suppressed UVB‐induced extracellular signal‐regulated kinase 2 (ERK2) activity and subsequently attenuated UVB‐induced phosphorylation of p90RSK by competitively binding with ATP. Constitutively active MEK (CA‐MEK) increased FRA1 phosphorylation and expression and also induced MMP‐1 expression, whereas dominant‐negative ERK2 (DN‐ERK2) had opposite effects. U0126, a MEK inhibitor, also decreased FRA1 phosphorylation and expression as well as MMP‐1 expression. The photoaging data obtained from mice clearly demonstrated that naringenin significantly inhibited UVB‐induced wrinkle formation, trans‐epidermal water loss and MMP‐13 expression. Naringenin exerts potent anti‐photoaging effects by suppressing ERK2 activity and decreasing FRA1 stability, followed by down‐regulation of AP‐1 transactivation and MMP‐1 expression.

Up-Regulation of Human Inducible Nitric Oxide Synthase by p300 Transcriptional Complex.

p300, a ubiquitous transcription coactivator, plays an important role in gene activation. Our previous work demonstrated that human inducible nitric oxide synthase (hiNOS) expression can be highly induced with the cytokine mixture (CM) of TNF-α + IL-1β + IFN-γ. In this study, we investigated the functional role of p300 in the regulation of hiNOS gene expression. Our initial data showed that overexpression of p300 significantly increased the basal and cytokine-induced hiNOS promoter activities in A549 cells. Interestingly, p300 activated cytokine-induced hiNOS transcriptional activity was completely abrogated by deleting the upstream hiNOS enhancer at -5 kb to -6 kb in the promoter. Furthermore, p300 over-expression increased cytokine-induced transcriptional activity on a heterologous minimal TK promoter with the same hiNOS enhancer. Site-directed mutagenesis of the hiNOS AP-1 motifs revealed that an intact upstream (-5.3kb) AP-1 binding site was critical for p300 mediated cytokine-induced hiNOS transcription. Furthermore, our ChIP analysis demonstrated that p300 was binding to Jun D and Fra-2 proteins at -5.3 kb AP-1 binding site in vivo. Lastly, our 3C assay was able to detect a long DNA loop between the hiNOS enhancer and core promoter site, and ChIP loop assay confirmed that p300 binds to AP-1 and RNA pol II proteins. Overall, our results suggest that coactivator p300 mediates cytokine-induced hiNOS transactivation by forming a distant DNA loop between its enhancer and core promoter region.

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

Requirement of Fra proteins for communication channels between cells in the filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120

The filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 differentiates specialized cells, heterocysts, that fix atmospheric nitrogen and transfer the fixed nitrogen to adjacent vegetative cells. Reciprocally, vegetative cells transfer fixed carbon to heterocysts. Several routes have been described for metabolite exchange within the filament, one of which involves communicating channels that penetrate the septum between adjacent cells. Several fra gene mutants were isolated 25 y ago on the basis of their phenotypes: inability to fix nitrogen and fragmentation of filaments upon transfer from N+ to N- media. Cryopreservation combined with electron tomography were used to investigate the role of three fra gene products in channel formation. FraC and FraG are clearly involved in channel formation, whereas FraD has a minor part. Additionally, FraG was located close to the cytoplasmic membrane and in the heterocyst neck, using immunogold labeling with antibody raised to the N-terminal domain of the FraG protein.

A cascade of iron-containing proteins governs the genetic iron starvation response to promote iron uptake and inhibit iron storage in fission yeast.

Iron is an essential cofactor, but it is also toxic at high levels. In Schizosaccharomyces pombe, the sensor glutaredoxin Grx4 guides the activity of the repressors Php4 and Fep1 to mediate a complex transcriptional response to iron deprivation: activation of Php4 and inactivation of Fep1 leads to inhibition of iron usage/storage, and to promotion of iron import, respectively. However, the molecular events ruling the activity of this double-branched pathway remained elusive. We show here that Grx4 incorporates a glutathione-containing iron-sulfur cluster, alone or forming a heterodimer with the BolA-like protein Fra2. Our genetic study demonstrates that Grx4-Fra2, but not Fep1 nor Php4, participates not only in iron starvation signaling but also in iron-related aerobic metabolism. Iron-containing Grx4 binds and inactivates the Php4 repressor; upon iron deprivation, the cluster in Grx4 is probably disassembled, the proteins dissociate, and Php4 accumulates at the nucleus and represses iron consumption genes. Fep1 is also an iron-containing protein, and the tightly bound iron is required for transcriptional repression. Our data suggest that the cluster-containing Grx4-Fra2 heterodimer constitutively binds to Fep1, and upon iron deprivation the disassembly of the iron cluster between Grx4 and Fra2 promotes reverse metal transfer from Fep1 to Grx4-Fra2, and de-repression of iron-import genes. Our genetic and biochemical study demonstrates that the glutaredoxin Grx4 independently governs the Php4 and Fep1 repressors through metal transfer. Whereas iron loss from Grx4 seems to be sufficient to release Php4 and allow its nuclear accumulation, total or partial disassembly of the Grx4-Fra2 cluster actively participates in iron-containing Fep1 activation by sequestering its iron and decreasing its interaction with promoters.

Signaling pathway STAT1 is strongly activated by IFN-β in the pathogenesis of osteoporosis.

BackgroundDespite extensive research, the underlying pathological mechanisms of osteoporosis are not completely understood. Recent studies have indicated a distinct role for the IFN-β/STAT1 pathway in bone metabolism. An inhibitory effect of IFN-β on osteoclastogenesis has been detected and STAT1/2 has been shown to influence osteoblastic bone metabolism. So far, no data concerning the IFN-β/STAT1 pathways in osteoblasts and osteoclasts from osteoporotic and non-osteoporotic patients are available. The aim of the study was to analyze these pathways in both cell types.MethodsOsteoblasts were isolated from the femoral heads of 12 osteoporotic and 11 non-osteoporotic patients and monocytes were differentiated into osteoclasts. After the differentiation period, cells were stimulated once with 20 and 100 ng/mL IFN-β for 4 days. Viability, activity, bone metabolism-related genes, and the proteins Fra1, SOCS1, STAT1, p-STAT1, and TRAF6 were analyzed.ResultsViability, activity, and gene expressions were not affected by stimulating the osteoblasts. However, in osteoporotic osteoclasts, which display a significantly higher basal osteoclastic activity, the stimulation with IFN-β lead to significant inhibition. Further, an increased STAT1 activation was detected in both cell types with no significant differences between the groups. Regarding the phosphorylation of STAT1, no significant influence was detected in osteoblasts but the IFN-β stimulation led to a significant increase of p-STAT1 in osteoclasts of both groups.ConclusionsIFN-β is a principal mediator in the pathogenesis of osteoporosis by inhibiting osteoclasts and inducing and activating STAT1. Our results also confirm this in cells from osteoporotic and non-osteoporotic patients. Strong inhibitory effects on the osteoclastogenesis of osteoporotic osteoclasts were detectable. Nevertheless, osteoblast activity was not negatively affected by IFN-β stimulation. These results may contribute to a better understanding of the underlying pathological signaling pathways of osteoporosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s40001-014-0074-4) contains supplementary material, which is available to authorized users.

Assessment of Folate Receptor-α and Epidermal Growth Factor Receptor Expression in Pemetrexed-Treated Non–Small-Cell Lung Cancer Patients

IntroductionFolate receptor-α regulates cellular uptake of folates and antifolates (eg, pemetrexed) and is frequently expressed in pulmonary adenocarcinoma. EGFR is an established therapeutic target in NSCLC. Therapies targeting FRA or EGFR are available. The association between FRA and EGFR expression in advanced NSCLC has not been explored. Combining therapeutic FRA antibodies with an EGFR inhibitor might be beneficial, if both of the targets are significantly coexpressed. Patients and MethodsSpecimens from 160 advanced NSCLC patients receiving pemetrexed-based chemotherapy were assessed for membranous FRA and EGFR protein expression using immunohistochemistry and the Hybrid (H)-score. EGFR (exons 18-21) and Kirsten RNA-associated rat sarcoma 2 virus (exon 2) mutations were determined. Results were correlated to patients' clinicopathological data, progression-free survival (PFS), and overall survival (OS). ResultsForty-seven patients (29%) had tumors with strong FRA and EGFR expression, but no statistically significant correlation was seen between protein levels of FRA and EGFR. High membranous FRA expression (H-score ≥ 20) was associated with prolonged PFS (5.5 vs. 3.4 months; hazard ratio [HR], 0.6060; P = .0254) and improved OS (12.1 vs. 6.4 months; HR, 0.5726; P = .0076). ConclusionSurvival times are improved in NSCLC patients whose tumors show strong membranous FRA expression. No statistical correlation between membranous FRA and EGFR expression was demonstrated in advanced NSCLC, but 47 patients (29%) had higher expression of both of the receptors and could be suitable for combined targeted therapies.

Fra-1 regulation of Matrix Metallopeptidase-1 (MMP-1) in metastatic variants of MDA-MB-231 breast cancer cells

Matrix Metallopeptidase 1 (MMP-1) expression has repeatedly been correlated to tumorigenesis and metastasis. Yet, MMP-1 regulation in a metastatic context remains largely unknown. Here we confirm differential MMP-1 expression in mammary carcinoma cells with varied metastatic potentials. We show that MMP-1 expression is regulated by an AP-1 element in its promoter in highly metastatic MDA-MB-231 mammary carcinoma cell derivatives. Fra-1, an AP-1 family transcription factor, differentially binds this element in highly metastatic cells compared to low metastatic cells and is required for MMP-1 expression. Overexpression of Fra-1 also caused increased MMP-1 expression. Fra-1 mRNA levels are unchanged in the cell variants, however its protein levels are higher in the metastatic cells. While there was no change in Fra-1 protein degradation rates, protein synthesis of Fra-1 was increased in the metastatic cell variant. These results demonstrate that Fra-1 and MMP-1 levels are differentially regulated in metastatic cell variants at the level of Fra-1 protein translation. Consistent with the importance of Fra-1 for tumor growth, we found that Fra-1 overexpression was sufficient to increase cell motility and anchorage independent growth. These results suggest that increased Fra-1 translation is critical for regulation of MMP-1 and tumor cell metastasis.

Abstract B39: Role of hepatoma upregulated Protein (HURP) in prostate cancer tumorigenesis.

Abstract Hepatoma up-regulated protein (HURP) is a cell cycle-regulated and microtubule-associated protein. It functions as a Ran GTPase effector and is involved in the stabilization of the mitotic kinetochore fibers. The expression of this protein is elevated in different tumor types and associated with tumor progression and aggressiveness. Also, a significant elevation of HURP expression was reported in normal stem cells including haematopoietic stem cells, mesenchymal stem cells, and mouse embryonic stem cells but not in differentiated cells, an evidence for HURP as stemness maintenance factor. The aim of this study is to analyze the oncogenic potential of HURP in prostate cancer (PCa). We analyzed the expression level of HURP in PCa patients and found the transcripts of HURP were overexpressed in PCa. The elevated expression of HURP in PCa was found to be associated with higher Gleason score, systemic progression and poor prognosis. Furthermore, using a case-control cohort study we demonstrated the overall prognostic ability of HURP mRNA in advanced PCa, as tested in receiver operating characteristic analysis [AUC 0.74 (95% CL, 0.68 to 0.8)]. In addition, we analyzed the expression level of HURP protein in tumor tissue and in prostate benign and cancer cell lines; we found that the level of HURP protein was almost 5-fold higher (p&amp;lt;0.005) relative to control (cystoprostatectomy) tissue and in cancer cell lines compared to the benign cells. Overall, these data suggest a strong relationship between the elevated expression of HURP and the development and progression of PCa. Next, we set out a tetracyclin-regulated transient expression system model to study the effects of HURP overexpression in LNCaP cells. Data obtained from western blot revealed that HURP overexpression was associated with the phosphorylation of C-Jun-N-Terminal kinase (JNK) and the induction of both Fra1 and c-JUN proteins as well as with the suppression of pro-apoptotic protein Bcl-Xs, and p53 without influencing the negative regulator MDM2. Also, the overexpression of HURP in CaP cell lines was found to enhance the expression of the anti-apoptotic protein Bcl-2 without influencing the anti-apoptotic protein. Conversely, viral titer of shHURP was found to inhibit the basal phosphorylation of JNK, to suppress the basal level of cyclin A, to increase the degradation of the inhibitor of NF-κB, to decrease the expression of PKC. Furthermore, down regulation of HURP was found to enhance the expression of p53 and to abolish its negative regulator MDM2, suggesting a central role for p53 in the modulation of HURP-induced cell proliferation and tumor progression. Also, the overexpression of HURP promoted the expression of the stemness markers Oct3/4 and Nanog, an evidence for the involvement of HURP in the maintenance of stemness properties in PCa cells. This idea is supported by the reported role of Nanog and Oct3/4 in promoting PCa stem cell characteristics as well as prostate resistance to androgen deprivation, and by our results showing decreased p53, with demonstrated roles in reprogramming, dedifferentiation, self-renewal, and pluripotency. Overall, our data suggest a potential role of HURP in PCa tumorigenesis. In addition, these findings suggest that HURP's stemness properties can contribute to its role in the etiology of PCa. Support: DOD PC094680 (CRG), PCF Creativity Award (CRG). Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B39. Citation Format: Abdelouahid El-Khattouti, Tangeng Ma, James R. Kent, Ingrid Espinoza, Christian R. Gomez. Role of hepatoma upregulated Protein (HURP) in prostate cancer tumorigenesis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B39.

Fra-1 regulation of Matrix Metallopeptidase-1 (MMP-1) in metastatic variants of MDA-MB-231 breast cancer cells.

Matrix Metallopeptidase 1 (MMP-1) expression has repeatedly been correlated to tumorigenesis and metastasis. Yet, MMP-1 regulation in a metastatic context remains largely unknown. Here we confirm differential MMP-1 expression in mammary carcinoma cells with varied metastatic potentials. We show that MMP-1 expression is regulated by an AP-1 element in its promoter in highly metastatic MDA-MB-231 mammary carcinoma cell derivatives. Fra-1, an AP-1 family transcription factor, differentially binds this element in highly metastatic cells compared to low metastatic cells and is required for MMP-1 expression. Overexpression of Fra-1 also caused increased MMP-1 expression. Fra-1 mRNA levels are unchanged in the cell variants, however its protein levels are higher in the metastatic cells. While there was no change in Fra-1 protein degradation rates, protein synthesis of Fra-1 was increased in the metastatic cell variant. These results demonstrate that Fra-1 and MMP-1 levels are differentially regulated in metastatic cell variants at the level of Fra-1 protein translation. Consistent with the importance of Fra-1 for tumor growth, we found that Fra-1 overexpression was sufficient to increase cell motility and anchorage independent growth. These results suggest that increased Fra-1 translation is critical for regulation of MMP-1 and tumor cell metastasis.

Signal-dependent fra-2 regulation in skeletal muscle reserve and satellite cells

Activator protein-1 (AP-1) is a ubiquitous transcription factor that paradoxically also has some tissue-specific functions. In skeletal muscle cells, we document that the AP-1 subunit, Fra-2, is expressed in the resident stem cells (Pax7-positive satellite cells) and also in the analogous undifferentiated ‘reserve' cell population in myogenic cultures, but not in differentiated myofiber nuclei. Silencing of Fra-2 expression enhances the expression of differentiation markers such as muscle creatine kinase and myosin heavy chain, indicating a possible role of Fra-2 in undifferentiated myogenic progenitor cells. We observed that Fra-2 is a target of cytokine-mediated extracellular signal-regulated kinase-1/2 signaling in cultured muscle cells, and extensive mass spectrometry and mutational analysis identified S320 and T322 as regulators of Fra-2 protein stability. Interestingly, Fra-2 S320 phosphorylation occurs transiently in activated satellite cells and is extinguished in myogenin-positive differentiating cells. Thus, cytokine-mediated Fra-2 expression and stabilization is linked to regulation of myogenic progenitor cells having implications for the molecular regulation of adult muscle stem cells and skeletal muscle regeneration.

Effect of estrogen and tamoxifen on the expression pattern of AP-1 factors in MCF-7 cells: role of c-Jun, c-Fos, and Fra-1 in cell cycle regulation

The activated transcription factor ERα plays an important role in the breast development and progression of cancer. In a non-classical pathway ER interacts with other transcription factors AP-1, NFkB, SP1, etc. AP-1 transcription factors control rapid responses of mammalian cells to stimuli that impact proliferation, differentiation, and transformation. AP-1 factors are leucine zipper proteins belonging to members of the Jun family (c-Jun, JunB, and JunD) and Fos family (c-Fos, FosB, Fra-1, and Fra-2) proteins. Although AP-1 factors are well characterized, not much is known about the expression pattern of the AP-1 factors in breast cancer cells. Hence to determine which AP-1 factors are expressed and regulated by estrogen, we used human breast cancer MCF-7 cells as in vitro model system. The MCF-7 cells were treated with or without estradiol-17β (E2) or antiestrogen tamoxifen (TMX) and the cell proliferation and viability was assessed by MTT assay. The expression of different AP-1 factors was analyzed by semi-quantitative RT-PCR. The cells treated with E2 found to increase the cell proliferation by more than 35 % and TMX an antiestrogen decreased by 29 % compared to control. The E2 found to induce the expression of c-Jun, Fra-1, and c-Fos, while TMX decreased the expression. In addition TMX also decreased the mRNA levels of Jun-D and Fra-2. These results suggest that the AP-1 factors c-Jun, c-Fos, and Fra-1 may be involved in the proliferation and transformation of MCF-7 cells. E2 also found to induce cyclin D1 and cyclin E1 mRNA transcripts of cell cycle regulators while TMX significantly decreased compared to control. Further E2 induced the anti-apoptotic Bcl-2 and TMX decreased mRNA transcripts. The data presented here support the E2-ERα-mediated MCF-7 cell proliferation and confirms the role of AP-1 factors in cell cycle regulation.

Transcriptional regulation of the mouse CD11c promoter by AP-1 complex with JunD and Fra2 in dendritic cells

CD11c, a member of the β2 integrin family of adhesion molecule, is expressed on the surface of myeloid lineages and activated lymphoid cells and forms a heterodimeric receptor with CD18. We analyzed the mouse CD11c promoter structure to elucidate the transcriptional regulation in dendritic cells (DCs). By reporter assay, the −84/−65 region was identified to be essential for activity of the mouse CD11c promoter in the mouse bone marrow-derived (BM) DCs and monocyte cell line RAW264.7. An electrophoretic mobility shift assay using a number of antibodies against transcription factors revealed that the target region was recognized by a complex including JunD and Fra2, which are transcription factors belonging to the AP-1 family. The direct interaction of JunD and Fra2 with the CD11c promoter was further confirmed by a chromatin immunoprecipitation assay using CD11c-positive cells purified from BMDCs. Finally, mouse JunD and/or Fra2 siRNA was introduced into BMDCs to evaluate the involvement of these factors against CD11c transcription and found that Fra2 siRNA reduced cell surface expression level of CD11c. These results indicate that AP-1 composed with JunD and Fra2 protein plays a primary role in enhancing the transcription level of the CD11c gene in DC.