GDNF

Gene Summary

Gene:GDNF; glial cell derived neurotrophic factor
Aliases: ATF1, ATF2, HSCR3, HFB1-GDNF
Location:5p13.1-p12
Summary:This gene encodes a highly conserved neurotrophic factor. The recombinant form of this protein was shown to promote the survival and differentiation of dopaminergic neurons in culture, and was able to prevent apoptosis of motor neurons induced by axotomy. The encoded protein is processed to a mature secreted form that exists as a homodimer. The mature form of the protein is a ligand for the product of the RET (rearranged during transfection) protooncogene. Multiple transcript variants encoding different isoforms have been found for this gene. Mutations in this gene may be associated with Hirschsprung disease. [provided by RefSeq, Jun 2010]
Databases:OMIM, VEGA, HGNC, Ensembl, GeneCard, Gene
Protein:glial cell line-derived neurotrophic factor
HPRD
Source:NCBIAccessed: 06 August, 2015

Ontology:

What does this gene/protein do?
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Cancer Overview

Research Indicators

Publications Per Year (1990-2015)
Graph generated 06 August 2015 using data from PubMed using criteria.

Literature Analysis

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Tag cloud generated 06 August, 2015 using data from PubMed, MeSH and CancerIndex

Specific Cancers (5)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: GDNF (cancer-related)

Thway K, Fisher C
Angiomatoid fibrous histiocytoma: the current status of pathology and genetics.
Arch Pathol Lab Med. 2015; 139(5):674-82 [PubMed] Related Publications
CONTEXT: Angiomatoid fibrous histiocytoma (AFH) is a rare soft tissue neoplasm of intermediate biologic potential and uncertain differentiation, most often arising in the superficial extremities of children and young adults. While it has characteristic histologic features of nodular distributions of ovoid and spindle cells with blood-filled cystic cavities and a surrounding dense lymphoplasmacytic infiltrate, there is a significant morphologic spectrum, which coupled with its rarity and lack of specific immunoprofile can make diagnosis challenging. Angiomatoid fibrous histiocytoma is associated with 3 characteristic gene fusions, EWSR1-CREB1 and EWSR1-ATF1, which are also described in other neoplasms, and rarely FUS-ATF1. Angiomatoid fibrous histiocytoma is now recognized at an increasing number of sites and is known to display a variety of unusual histologic features.
OBJECTIVE: To review the current status of AFH, discussing putative etiology, histopathology with variant morphology and differential diagnosis, and current genetics, including overlap with other tumors harboring EWSR1-CREB1 and EWSR1-ATF1 fusions.
DATA SOURCES: Review of published literature, including case series, case reports, and review articles, in online medical databases.
CONCLUSIONS: The occurrence of AFH at several unusual anatomic sites and its spectrum of morphologic patterns can result in significant diagnostic difficulty, and correct diagnosis is particularly important because of its small risk of metastasis and death. This highlights the importance of diagnostic recognition, ancillary molecular genetic confirmation, and close clinical follow-up of patients with AFH. Further insight into the genetic and epigenetic changes arising secondary to the characteristic gene fusions of AFH will be integral to understanding its tumorigenic mechanisms.

Wang J, Thway K
Clear cell sarcoma-like tumor of the gastrointestinal tract: an evolving entity.
Arch Pathol Lab Med. 2015; 139(3):407-12 [PubMed] Related Publications
Clear cell sarcoma-like tumor of the gastrointestinal tract (CCSLGT) is a rare malignant neoplasm that occurs in the wall of the small bowel, stomach, or large bowel, predominantly in young adults. It is an aggressive neoplasm that frequently presents with metastatic disease and has a high mortality rate. Histologically, it is usually composed of medium-sized primitive ovoid or epithelioid cells with pale or clear cytoplasm that are arranged in sheets or in papillary or alveolar architectures. Clear cell sarcoma-like tumor of the gastrointestinal tract is positive for S100 protein, invariably negative for melanocyte-specific markers and is often also positive for neuroendocrine markers. The etiology of CCSLGT is unknown, but many studies have shown associations with EWSR1-CREB1 gene fusions and, less frequently, with EWSR1-ATF1 fusions. Here, we discuss the current status of CCSLGT, including histologic, immunophenotypic, and molecular findings.

Nam EH, Lee Y, Moon B, et al.
Twist1 and AP-1 cooperatively upregulate integrin α5 expression to induce invasion and the epithelial-mesenchymal transition.
Carcinogenesis. 2015; 36(3):327-37 [PubMed] Related Publications
Epithelial-mesenchymal transition (EMT) is an important process implicated in tumor invasion and metastasis. Twist1 is a transcription factor that induces EMT, including E-cadherin suppression and cancer cell migration and invasion; hence it promotes cancer metastasis. Twist1 directly or indirectly regulates the expression of various genes and cellular functions involved in cancer progression. However, the underlying mechanisms remain largely unknown. In this study, we investigated the molecular basis for Twist1-mediated invasion and EMT. In human cancer cells, Twist1 was found to directly upregulate transcription of the mesenchymal gene integrin α5 in an E-box-independent, but activating protein-1 (AP-1) element-dependent, manner. Twist1 activated the integrin α5 promoter by interacting with and activating the transcription factor AP-1, composed of c-Jun and activating transcription factor-2 (ATF-2); it also enhanced the nuclear presence of ATF-2. AP-1 was critical for Twist1-induced cancer cell invasion, primarily through the induction of integrin α5, which activated c-Jun N-terminal kinase and focal adhesion kinase-signaling activities. Using data from The Cancer Genome Atlas, we found that Twist1 expression positively correlates with integrin α5 expression in human colorectal cancers. These findings suggest that cooperation between Twist1 and AP-1 represents a novel mechanism for EMT and tumor invasiveness. This study supports further investigation into the molecular basis underlying the diverse Twist1-mediated functions that occur during tumor progression.

García JJ, Jin L, Jackson SB, et al.
Primary pulmonary hyalinizing clear cell carcinoma of bronchial submucosal gland origin.
Hum Pathol. 2015; 46(3):471-5 [PubMed] Related Publications
Hyalinizing clear cell carcinoma (HCCC) has only been described in salivary glands of the head and neck. We report a 38-year-old man with a 2.6-cm lung tumor that was growing in a peribronchial location and had morphologic features of HCCC. The tumor cells expressed cytokeratin 7 and keratin AE1/AE3, and the vast majority of tumor cells marked also with p63 and p40. They were negative for cytokeratin 20, S-100, smooth muscle actin, napsin A, and thyroid transcription factor-1. Fluorescence in situ hybridization revealed Ewing Sarcoma Breakpoint Region 1 (EWSR1) rearrangement, and reverse-transcription polymerase chain reaction confirmed the presence of the EWSR1-Activating Transcription Factor 1 (ATF1) fusion transcript, which was subsequently sequenced. The morphologic, immunophenotypic, cytogenetic, and molecular findings together with the patient's history and location of the tumor support a diagnosis of primary pulmonary HCCC of bronchial submucosal gland origin. It is our understanding that this is the first report of HCCC arising as a primary tumor outside the head and neck region.

Gozdecka M, Lyons S, Kondo S, et al.
JNK suppresses tumor formation via a gene-expression program mediated by ATF2.
Cell Rep. 2014; 9(4):1361-74 [PubMed] Related Publications
JNK and p38 phosphorylate a diverse set of substrates and, consequently, can act in a context-dependent manner to either promote or inhibit tumor growth. Elucidating the functions of specific substrates of JNK and p38 is therefore critical for our understanding of these kinases in cancer. ATF2 is a phosphorylation-dependent transcription factor and substrate of both JNK and p38. Here, we show ATF2 suppresses tumor formation in an orthotopic model of liver cancer and cellular transformation in vitro. Furthermore, we find that suppression of tumorigenesis by JNK requires ATF2. We identify a transcriptional program activated by JNK via ATF2 and provide examples of JNK- and ATF2-dependent genes that block cellular transformation. Significantly, we also show that ATF2-dependent gene expression is frequently downregulated in human cancers, indicating that amelioration of JNK-ATF2-mediated suppression may be a common event during tumor development.

Zheng R, Wang X, Studzinski GP
1,25-Dihydroxyvitamin D3 induces monocytic differentiation of human myeloid leukemia cells by regulating C/EBPβ expression through MEF2C.
J Steroid Biochem Mol Biol. 2015; 148:132-7 [PubMed] Article available free on PMC after 01/04/2016 Related Publications
Myogenic enhancer factor2 (Mef2) consists of a family of transcription factors involved in morphogenesis of skeletal, cardiac and smooth muscle cells. Among the four isoforms (Mef2A, 2B, 2C, and 2D), Mef2C was also found to play important roles in hematopoiesis. At myeloid progenitor level, Mef2C expression favors monocytic differentiation. Previous studies from our laboratory demonstrated that ERK5 was activated in 1,25-dihydroxyvitamin D3 (1,25D)-induced monocytic differentiation in AML cells and ERK5 activation was accompanied by increased Mef2C phosphorylation. We therefore examined the role of Mef2C in 1,25D-induced monocytic differentiation in AML cell lines (HL60, U937 and THP1) and found that knockdown of Mef2C with small interfering RNA (siRNA) significantly decreases the expression of the monocytic marker, CD14, without affecting the expression of the general myeloid marker, CD11b. CCAAT/enhancer-binding protein (C/EBP) β, which can bind to CD14 promoter and increase its transcription, has been shown to be the downstream effector of 1,25D-induced monocytic differentiation in AML cells. When Mef2C was knocked down, expression of C/EBPβ was reduced at both mRNA and protein levels. The protein expression levels of cell cycle regulators, p27(Kip1) and cyclin D1, were not affected by Mef2C knockdown, nor the monopoiesis related transcription factor, ATF2 (activating transcription factor 2). Thus, we conclude that 1,25D-induced monocytic differentiation, and CD14 expression in particular, are mediated through activation of ERK5-Mef2C-C/EBPβ signaling pathway, and that Mef2C does not seem to modulate cell cycle progression.

Nakano T, Yamamoto H, Nishijima T, et al.
Hyalinizing clear cell carcinoma with EWSR1-ATF1 fusion gene: report of three cases with molecular analyses.
Virchows Arch. 2015; 466(1):37-43 [PubMed] Related Publications
Hyalinizing clear cell carcinoma (HCCC) is a low-grade salivary gland carcinoma characterized by clear cells and hyalinized stroma. Recently, the EWSR1-ATF1 fusion gene was found in HCCCs. We herein describe three cases of HCCC identified in one male and two females, ranging in age from 27 to 67 years. The tumors were located in the root of tongue, nasopharynx, and soft palate. They were composed of nested or cord-like proliferations of epithelial cells with clear to pale eosinophilic cytoplasm, embedded in hyalinized and focally fibroedematous stroma. Tumor-associated lymphoid proliferation and pseudoepitheliomatous hyperplasia were also observed in each one case. MAML2 fusions specific to mucoepidermoid carcinoma were not detected in any of the three cases. We found EWSR1-ATF1 in two of three HCCCs using reverse transcription polymerase chain reaction (RT-PCR) with our original primer sets designed to detect the fusion gene transcripts in formalin-fixed paraffin-embedded (FFPE) tissues. EWSR1 rearrangement was also confirmed by fluorescence in situ hybridization (FISH) on FFPE sections in two cases. There was a good concordance between the two methods (two positive cases and one negative case by both RT-PCR and FISH). Therefore, RT-PCR and FISH using FFPE tissue may be ancillary tools to confirm the diagnosis of HCCC.

Lo Iacono M, Monica V, Vavalà T, et al.
ATF2 contributes to cisplatin resistance in non-small cell lung cancer and celastrol induces cisplatin resensitization through inhibition of JNK/ATF2 pathway.
Int J Cancer. 2015; 136(11):2598-609 [PubMed] Related Publications
ATF2 is a transcription factor involved in stress and DNA damage. A correlation between ATF2 JNK-mediated activation and resistance to damaging agents has already been reported. The purpose of the present study was to investigate whether ATF2 may have a role in acquired resistance to cisplatin in non-small cell lung cancer (NSCLC). mRNA and protein analysis on matched cancer and corresponding normal tissues from surgically resected NSCLC have been performed. Furthermore, in NSCLC cell lines, ATF2 expression levels were evaluated and correlated to platinum (CDDP) resistance. Celastrol-mediated ATF2/cJUN activity was measured. High expression levels of both ATF2 transcript and proteins were observed in lung cancer specimens (p < 0.01, Log2 (FC) = +4.7). CDDP-resistant NSCLC cell lines expressed high levels of ATF2 protein. By contrast, Celastrol-mediated ATF2/cJUN functional inhibition restored the response to CDDP. Moreover, ATF2 protein activation correlates with worse outcome in advanced CDDP-treated patients. For the first time, it has been shown NSCLC ATF2 upregulation at both mRNA/protein levels in NSCLC. In addition, we reported that in NSCLC cell lines a correlation between ATF2 protein expression and CDDP resistance occurs. Altogether, our results indicate a potential increase in CDDP sensitivity, on Celastrol-mediated ATF2/cJUN inhibition. These data suggest a possible involvement of ATF2 in NSCLC CDDP-resistance.

Pletneva MA, Andea A, Palanisamy N, et al.
Clear cell melanoma: a cutaneous clear cell malignancy.
Arch Pathol Lab Med. 2014; 138(10):1328-36 [PubMed] Related Publications
Clear cell melanoma is a rare clear cell malignancy. Accurate diagnosis of clear cell melanoma requires integration of immunohistochemical and morphologic findings, with molecular studies to rule out clear cell sarcoma. The differential diagnosis includes melanoma, carcinoma, perivascular epithelioid cell tumor, and epidermotropic clear cell sarcoma. We use a case of a lesion on the helix of an 86-year-old man as an example. Histologic examination revealed an ulcerated clear cell malignant tumor. Tumor cell cytoplasm contained periodic acid-Schiff-positive, diastase-sensitive glycogen. Tumor cells showed positive labeling for S100, HMB-45, and Melan-A, and negative labeling for cytokeratins, p63, and smooth muscle actin. Molecular studies demonstrated BRAF V600E mutation, copy gains at the 6p25 (RREB1) and 11q13 (CCND1) loci, and absence of EWSR1-ATF1 fusion. These findings supported a diagnosis of clear cell melanoma. The rare pure clear cell morphology occurs due to accumulation of intracytoplasmic glycogen. We review the differential diagnosis of clear cell melanoma and describe the utility of immunohistochemical and molecular studies in confirming this diagnosis.

Agaram NP, Chen HW, Zhang L, et al.
EWSR1-PBX3: a novel gene fusion in myoepithelial tumors.
Genes Chromosomes Cancer. 2015; 54(2):63-71 [PubMed] Article available free on PMC after 01/04/2016 Related Publications
The genetics of myoepithelial tumors (ME) of soft tissue and bone have recently been investigated, with EWSR1-related gene fusions being seen in approximately half of the tumors. The fusion partners of EWSR1 so far described include POU5F1, PBX1, ZNF444 and, in a rare case, ATF1. We investigated by RNA sequencing an index case of EWSR1-rearranged ME of the tibia, lacking a known fusion partner, and identified a novel EWSR1-PBX3 fusion. The fusion was further validated by reverse transcriptase polymerase chain reaction and fluorescence in situ hybridization (FISH). To evaluate if this is a recurrent event, an additional cohort of 22 EWSR1-rearranged ME cases lacking a fusion partner were screened by FISH for abnormalities in PBX3 gene. Thus, two additional cases were identified showing an EWSR1-PBX3 gene fusion. One of them was also intraosseous involving the ankle, while the other occurred in the soft tissue of the index finger. The morphology of the EWSR1-PBX3 fusion positive cases showed similar findings, with nests or sheets of epithelioid to spindle cells in a partially myxoid to collagenous matrix. All three cases showed expression of S100 and EMA by immunohistochemistry. In summary, we report a novel EWSR1-PBX3 gene fusion in a small subset of ME, thereby expanding the spectrum of EWSR1-related gene fusions seen in these tumors. This gene fusion seems to occur preferentially in skeletal ME, with two of the three study cases occurring in intraosseous locations.

Rudalska R, Dauch D, Longerich T, et al.
In vivo RNAi screening identifies a mechanism of sorafenib resistance in liver cancer.
Nat Med. 2014; 20(10):1138-46 [PubMed] Related Publications
In solid tumors, resistance to therapy inevitably develops upon treatment with cytotoxic drugs or molecularly targeted therapies. Here, we describe a system that enables pooled shRNA screening directly in mouse hepatocellular carcinomas (HCC) in vivo to identify genes likely to be involved in therapy resistance. Using a focused shRNA library targeting genes located within focal genomic amplifications of human HCC, we screened for genes whose inhibition increased the therapeutic efficacy of the multikinase inhibitor sorafenib. Both shRNA-mediated and pharmacological silencing of Mapk14 (p38α) were found to sensitize mouse HCC to sorafenib therapy and prolong survival by abrogating Mapk14-dependent activation of Mek-Erk and Atf2 signaling. Elevated Mapk14-Atf2 signaling predicted poor response to sorafenib therapy in human HCC, and sorafenib resistance of p-Mapk14-expressing HCC cells could be reverted by silencing Mapk14. Our results suggest that a combination of sorafenib and Mapk14 blockade is a promising approach to overcoming therapy resistance of human HCC.

Liu C, Ren Y, Li X, et al.
Absence of 19 known hotspot oncogenic mutations in soft tissue clear cell sarcoma: two cases report with review of the literature.
Int J Clin Exp Pathol. 2014; 7(8):5242-9 [PubMed] Article available free on PMC after 01/04/2016 Related Publications
Clear cell sarcoma (CCS) of the tendons and aponeuroses is a rare soft tissue sarcoma that morphologically resembles cutaneous malignant melanoma but exhibits a distinct molecular profile. Gastrointestinal (GI) CCS is extremely rare. In this study, two cases of CCS were presented: (1) left thumb and (2) jejunum. Case 1 manifested the characteristic CCS morphology. Case 2 was morphologically unusual and difficult to diagnose. Immunohistochemically, the two cases of tumor cells were diffusely positive for S100, vimentin, NSE protein, focal expression of CgA, and CAM2.5 protein. In case 1, the tumor cells were diffusely positive for HMB45, focal expression of CD56, and melan A antigen. Reverse transcriptase-polymerase chain reaction (RT-PCR) results confirmed the presence of the EWS/ATF1 translocation (type 1) in the two cases. Then, we detected 19 hotspot oncogenes in the two cases. To the best of our knowledge, this study is the first to apply a high-throughput OncoCarta panel 1.0 and MassARRAY system to detect 238 known mutations in 19 hotspot oncogenes in soft tissue clear cell sarcoma. In this study, no mutations were observed in these hotspot oncogenes in the two cases.

Rudraraju B, Droog M, Abdel-Fatah TM, et al.
Phosphorylation of activating transcription factor-2 (ATF-2) within the activation domain is a key determinant of sensitivity to tamoxifen in breast cancer.
Breast Cancer Res Treat. 2014; 147(2):295-309 [PubMed] Related Publications
Activating transcription factor-2 (ATF-2) has been implicated as a tumour suppressor in breast cancer (BC). c-JUN N-terminal kinase (JNK) and p38 MAPK phosphorylate ATF-2 within the activation domain (AD), which is required for its transcriptional activity. To date, the role of ATF-2 in determining response to endocrine therapy has not been explored. Effects of ATF-2 loss in the oestrogen receptor (ER)-positive luminal BC cell line MCF7 were explored, as well as its role in response to tamoxifen treatment. Genome-wide chromatin binding patterns of ATF-2 when phosphorylated within the AD in MCF-7 cells were determined using ChIP-seq. The expression of ATF-2 and phosphorylated ATF-2 (pATF-2-Thr71) was determined in a series of 1,650 BC patients and correlated with clinico-pathological features and clinical outcome. Loss of ATF-2 diminished the growth-inhibitory effects of tamoxifen, while tamoxifen treatment induced ATF-2 phosphorylation within the AD, to regulate the expression of a set of 227 genes for proximal phospho-ATF-2 binding, involved in cell development, assembly and survival. Low expression of both ATF-2 and pATF-2-Thr71 was significantly associated with aggressive pathological features. Furthermore, pATF-2 was associated with both p-p38 and pJNK1/2 (< 0.0001). While expression of ATF-2 is not associated with outcome, pATF-2 is associated with longer disease-free (p = 0.002) and BC-specific survival in patients exposed to tamoxifen (p = 0.01). Furthermore, multivariate analysis confirmed pATF-2-Thr71 as an independent prognostic factor. ATF-2 is important for modulating the effect of tamoxifen and phosphorylation of ATF-2 within the AD at Thr71 predicts for improved outcome for ER-positive BC receiving tamoxifen.

Diaz-Rodriguez E, Garcia-Rendueles AR, Ibáñez-Costa A, et al.
Somatotropinomas, but not nonfunctioning pituitary adenomas, maintain a functional apoptotic RET/Pit1/ARF/p53 pathway that is blocked by excess GDNF.
Endocrinology. 2014; 155(11):4329-40 [PubMed] Related Publications
Acromegaly is caused by somatotroph cell adenomas (somatotropinomas [ACROs]), which secrete GH. Human and rodent somatotroph cells express the RET receptor. In rodents, when normal somatotrophs are deprived of the RET ligand, GDNF (Glial Cell Derived Neurotrophic Factor), RET is processed intracellularly to induce overexpression of Pit1 [Transcription factor (gene : POUF1) essential for transcription of Pituitary hormones GH, PRL and TSHb], which in turn leads to p19Arf/p53-dependent apoptosis. Our purpose was to ascertain whether human ACROs maintain the RET/Pit1/p14ARF/p53/apoptosis pathway, relative to nonfunctioning pituitary adenomas (NFPAs). Apoptosis in the absence and presence of GDNF was studied in primary cultures of 8 ACROs and 3 NFPAs. Parallel protein extracts were analyzed for expression of RET, Pit1, p19Arf, p53, and phospho-Akt. When GDNF deprived, ACRO cells, but not NFPAs, presented marked level of apoptosis that was prevented in the presence of GDNF. Apoptosis was accompanied by RET processing, Pit1 accumulation, and p14ARF and p53 induction. GDNF prevented all these effects via activation of phospho-AKT. Overexpression of human Pit1 (hPit1) directly induced p19Arf/p53 and apoptosis in a pituitary cell line. Using in silico studies, 2 CCAAT/enhancer binding protein alpha (cEBPα) consensus-binding sites were found to be 100% conserved in mouse, rat, and hPit1 promoters. Deletion of 1 cEBPα site prevented the RET-induced increase in hPit1 promoter expression. TaqMan qRT-PCR (real time RT-PCR) for RET, Pit1, Arf, TP53, GDNF, steroidogenic factor 1, and GH was performed in RNA from whole ACRO and NFPA tumors. ACRO but not NFPA adenomas express RET and Pit1. GDNF expression in the tumors was positively correlated with RET and negatively correlated with p53. In conclusion, ACROs maintain an active RET/Pit1/p14Arf/p53/apoptosis pathway that is inhibited by GDNF. Disruption of GDNF's survival function might constitute a new therapeutic route in acromegaly.

Revilla S, Ursulet S, Álvarez-López MJ, et al.
Lenti-GDNF gene therapy protects against Alzheimer's disease-like neuropathology in 3xTg-AD mice and MC65 cells.
CNS Neurosci Ther. 2014; 20(11):961-72 [PubMed] Related Publications
AIMS: Glial cell-derived neurotrophic factor (GDNF) is emerging as a potent neurotrophic factor with therapeutic potential against a range of neurodegenerative conditions including Alzheimer's disease (AD). We assayed the effects of GDNF treatment in AD experimental models through gene-therapy procedures.
METHODS: Recombinant lentiviral vectors were used to overexpress GDNF gene in hippocampal astrocytes of 3xTg-AD mice in vivo, and also in the MC65 human neuroblastoma that conditionally overexpresses the 99-residue carboxyl-terminal (C99) fragment of the amyloid precursor protein.
RESULTS: After 6 months of overexpressing GDNF, 10-month-old 3xTg-AD mice showed preserved learning and memory, while their counterparts transduced with a green fluorescent protein vector showed cognitive loss. GDNF therapy did not significantly reduce amyloid and tau pathology, but rather, induced a potent upregulation of brain-derived neurotrophic factor that may act in concert with GDNF to protect neurons from atrophy and degeneration. MC65 cells overexpressing GDNF showed an abolishment of oxidative stress and cell death that was at least partially mediated by a reduced presence of intracellular C99 and derived amyloid β oligomers.
CONCLUSIONS: GDNF induced neuroprotection in the AD experimental models used. Lentiviral vectors engineered to overexpress GDNF showed to be safe and effective, both as a potential gene therapy and as a tool to uncover the mechanisms of GDNF neuroprotection, including cross talk between astrocytes and neurons in the injured brain.

Xiao N, Lin Y, Cao H, et al.
Neurotrophic factor GDNF promotes survival of salivary stem cells.
J Clin Invest. 2014; 124(8):3364-77 [PubMed] Article available free on PMC after 01/04/2016 Related Publications
Stem cell-based regenerative therapy is a promising treatment for head and neck cancer patients that suffer from chronic dry mouth (xerostomia) due to salivary gland injury from radiation therapy. Current xerostomia therapies only provide temporary symptom relief, while permanent restoration of salivary function is not currently feasible. Here, we identified and characterized a stem cell population from adult murine submandibular glands. Of the different cells isolated from the submandibular gland, this specific population, Lin-CD24+c-Kit+Sca1+, possessed the highest capacity for proliferation, self renewal, and differentiation during serial passage in vitro. Serial transplantations of this stem cell population into the submandibular gland of irradiated mice successfully restored saliva secretion and increased the number of functional acini. Gene-expression analysis revealed that glial cell line-derived neurotrophic factor (Gdnf) is highly expressed in Lin-CD24+c-Kit+Sca1+ stem cells. Furthermore, GDNF expression was upregulated upon radiation therapy in submandibular glands of both mice and humans. Administration of GDNF improved saliva production and enriched the number of functional acini in submandibular glands of irradiated animals and enhanced salisphere formation in cultured salivary stem cells, but did not accelerate growth of head and neck cancer cells. These data indicate that modulation of the GDNF pathway may have potential therapeutic benefit for management of radiation-induced xerostomia.

Liu Z, Zhang J, Gao Y, et al.
Large-scale characterization of DNA methylation changes in human gastric carcinomas with and without metastasis.
Clin Cancer Res. 2014; 20(17):4598-612 [PubMed] Article available free on PMC after 01/09/2015 Related Publications
PURPOSE: Metastasis is the leading cause of death for gastric carcinoma. An epigenetic biomarker panel for predicting gastric carcinoma metastasis could have significant clinical impact on the care of patients with gastric carcinoma. The main purpose of this study is to characterize the methylation differences between gastric carcinomas with and without metastasis.
EXPERIMENTAL DESIGN: Genome-wide DNA methylation profiles between 4 metastatic and 4 nonmetastatic gastric carcinomas and their surgical margins (SM) were analyzed using methylated-CpG island amplification with microarray. The methylation states of 73 candidate genes were further analyzed in patients with gastric carcinoma in a discovery cohort (n=108) using denatured high performance liquid chromatography, bisulfite-sequencing, and MethyLight. The predictive values of potential metastasis-methylation biomarkers were validated in cohorts of patients with gastric carcinoma in China (n=330), Japan (n=129), and Korea (n=153).
RESULTS: The gastric carcinoma genome showed significantly higher proportions of hypomethylation in the promoter and exon-1 regions, as well as increased hypermethylation of intragenic fragments when compared with SMs. Significant differential methylation was validated in the CpG islands of 15 genes (P<0.05) and confirmed using bisulfite sequencing. These genes included BMP3, BNIP3, CDKN2A, ECEL1, ELK1, GFRA1, HOXD10, KCNH1, PSMD10, PTPRT, SIGIRR, SRF, TBX5, TFPI2, and ZNF382. Methylation changes of GFRA1, SRF, and ZNF382 resulted in up- or downregulation of their transcription. Most importantly, the prevalence of GFRA1, SRF, and ZNF382 methylation alterations was consistently and coordinately associated with gastric carcinoma metastasis and the patients' overall survival throughout discovery and validation cohorts in China, Japan, and Korea.
CONCLUSION: Methylation changes of GFRA1, SRF, and ZNF382 may be a potential biomarker set for prediction of gastric carcinoma metastasis.

Chen SS, Yang C, Hao F, et al.
Intrastriatal GDNF gene transfer by inducible lentivirus vectors protects dopaminergic neurons in a rat model of parkinsonism.
Exp Neurol. 2014; 261:87-96 [PubMed] Related Publications
Glial cell line-derived neurotrophic factor (GDNF) has neuroprotective effects on dopaminergic (DA) neurons both in vivo and in vitro. However, substantial evidence has shown that a long-term overexpression of GDNF gene is often associated with side effects. We previously improved tetracycline (Tet)-On lentivirus system carrying human GDNF (hGDNF) gene, and demonstrated that hGDNF gene expression was tightly regulated and functional in vitro. Here we further examined the efficiency and neuroprotection of Tet-On lentivirus-mediated hGDNF gene regulation in neural progenitor cells (NPCs) and a rat model of parkinsonism. The results showed that hGDNF gene expression was tightly regulated in transduced NPCs. Doxycycline (Dox)-induced hGDNF protected DA neurons from 6-hydroxydopamine (6-OHDA)-induced toxicity in vitro. Intrastriatal injections of Tet-On lentivirus vectors resulted in dramatically increased levels of hGDNF protein in the striatum of rats with Dox-drinking water, when compared to lentivirus-injected and saline-injected rats with normal drinking water, respectively. In addition, hGDNF protected nigral DA neurons and striatal DA fibers, and attenuated d-amphetamine-induced rotational asymmetry in the 6-OHDA lesioned rats. To the best of our knowledge, this is the first report that hGDNF gene transfer by Tet-On lentivirus vectors is tightly regulated in rat brain, and Dox-induced hGDNF is functional in neuroprotection of nigral DA neurons in a rat model of parkinsonism.

Yang M, Kim HS, Cho MY
Different methylation profiles between intestinal and diffuse sporadic gastric carcinogenesis.
Clin Res Hepatol Gastroenterol. 2014; 38(5):613-20 [PubMed] Related Publications
OBJECTIVE: Gastric cancer (GC) is histologically classified into intestinal type and diffuse type, and diffuse type cancer can be further subdivided into poorly differentiated carcinoma (PDC) and signet ring cell carcinoma (SRCC). Recent evidence suggests that early SRCC is an initial, differentiated form of diffuse GC that may evolve into PDC. This study aimed at identifying the molecular features of epigenetic methylation changes in histologic differentiation status of GC.
METHODS: Included in this study are 149 samples of paraffin-embedded tissues and 115 fresh endoscopically biopsied tissues. Multiple paraffin tissues involving normal (n=22), dysplasias (GDs, n=39), differentiated cancers (DCs, n=35), PDCs (n=33) and SRCCs (n=20) were included as an experimental group. For the validation group, endoscopically biopsied tissues of DCs (n=50), PDCs (n=31), and SRCs (n=34) were analyzed. DNAs, isolated from each group were analyzed to determine the methylation status of 6 genes (GDNF, RORA, MINT25, KLF7, CDH1, LINE-1) using pyrosequencing.
RESULTS: LINE-1 was hypomethylated in GCs compared to normal and GD. GDNF, RORA and MINT25 were more hypermethylated in intestinal type GCs than those of diffuse type GCs, whereas CDH1 showed opposite patterns of methylation. Among diffuse type GCs, SRCCs showed lower level of methylation for GDNF, RORA, MINT25 and KLF7, and higher level for CDH1 compared to PDCs.
CONCLUSIONS: In conclusion, intestinal type of GCs shows different epigenetic methylation profiles compared to the diffuse one. Moreover, SRCCs have different methylation profiles compared with PDCs, suggesting a unique molecular pathway in the gastric carcinogenesis.

Outani H, Tanaka T, Wakamatsu T, et al.
Establishment of a novel clear cell sarcoma cell line (Hewga-CCS), and investigation of the antitumor effects of pazopanib on Hewga-CCS.
BMC Cancer. 2014; 14:455 [PubMed] Article available free on PMC after 01/09/2015 Related Publications
BACKGROUND: Clear cell sarcoma (CCS) is a therapeutically unresolved, aggressive, soft tissue sarcoma (STS) that predominantly affects young adults. This sarcoma is defined by t(12;22)(q13;q12) translocation, which leads to the fusion of Ewing sarcoma gene (EWS) to activating transcription factor 1 (ATF1) gene, producing a chimeric EWS-ATF1 fusion gene. We established a novel CCS cell line called Hewga-CCS and developed an orthotopic tumor xenograft model to enable comprehensive bench-side investigation for intensive basic and preclinical research in CCS with a paucity of experimental cell lines.
METHODS: Hewga-CCS was derived from skin metastatic lesions of a CCS developed in a 34-year-old female. The karyotype and chimeric transcript were analyzed. Xenografts were established and characterized by morphology and immunohistochemical reactivity. Subsequently, the antitumor effects of pazopanib, a recently approved, novel, multitargeted, tyrosine kinase inhibitor (TKI) used for the treatment of advanced soft tissue sarcoma, on Hewga-CCS were assessed in vitro and in vivo.
RESULTS: Hewga-CCS harbored the type 2 EWS-ATF1 transcript. Xenografts morphologically mimicked the primary tumor and expressed S-100 protein and antigens associated with melanin synthesis (Melan-A, HMB45). Pazopanib suppressed the growth of Hewga-CCS both in vivo and in vitro. A phospho-receptor tyrosine kinase array revealed phosphorylation of c-MET, but not of VEGFR, in Hewga-CCS. Subsequent experiments showed that pazopanib exerted antitumor effects through the inhibition of HGF/c-MET signaling.
CONCLUSIONS: CCS is a rare, devastating disease, and our established CCS cell line and xenograft model may be a useful tool for further in-depth investigation and understanding of the drug-sensitivity mechanism.

Rusmini M, Griseri P, Matera I, et al.
Expression variability and function of the RET gene in adult peripheral blood mononuclear cells.
J Cell Physiol. 2014; 229(12):2027-37 [PubMed] Related Publications
RET is a gene playing a key role during embryogenesis and in particular during the enteric nervous system development. High levels of RET gene expression are maintained in different human tissues also in adulthood, although their physiological role remains unclear. In particular, collected evidences of a RET contribution in the development and maintenance of the immune system prompted us to investigate its levels of surface expression on peripheral blood mononuclear cells (PBMCs) from adult healthy donors. Despite variability among samples, RET expression was conserved at similar levels in the different immune cell subsets, with higher correlations in similar lymphocyte populations (i.e. CD4(+) and CD8(+) T cells). Conversely, no correlation was found between the amount of RET receptor, the expression of its putative ligands and co-receptors and the genotypes at the RET locus. Moreover, we investigated the RET-associated inflammatory pathways in PBMCs from healthy donors both in resting conditions and upon glial cell derived neurotrophic factor (GDNF) and GPI-linked co-receptors alpha 1 (GFRα1) mediated RET activation. RET mRNA levels positively correlated with the transcript amount of interleukin-8 (IL-8), a cytokine produced by monocytes and macrophages, though we could not demonstrate its direct effect on RET expression by in vitro experiments on THP1 human monocytic cells. These results imply that RET expression might be influenced by either cis- and/or trans-factors, which together would account for its high variability within the general population, and suggest a putative functional role of the RET gene in modulating immune cell responses during inflammation and carcinogenesis.

Romanov VS, Brichkina AI, Morrison H, et al.
Novel mechanism of JNK pathway activation by adenoviral E1A.
Oncotarget. 2014; 5(8):2176-86 [PubMed] Article available free on PMC after 01/09/2015 Related Publications
The adenoviral oncoprotein E1A influences cellular regulation by interacting with a number of cellular proteins. In collaboration with complementary oncogenes, E1A fully transforms primary cells. As part of this action, E1A inhibits transcription of c-Jun:Fos target genes while promoting that of c-Jun:ATF2-dependent genes including jun. Both c-Jun and ATF2 are hyperphosphorylated in response to E1A. In the current study, E1A was fused with the ligand binding domain of the estrogen receptor (E1A-ER) to monitor the immediate effect of E1A activation. With this approach we now show that E1A activates c-Jun N-terminal kinase (JNK), the upstream kinases MKK4 and MKK7, as well as the small GTPase Rac1. Activation of the JNK pathway requires the N-terminal domain of E1A, and, importantly, is independent of transcription. In addition, it requires the presence of ERM proteins. Downregulation of signaling components upstream of JNK inhibits E1A-dependent JNK/c-Jun activation. Taking these findings together, we show that E1A activates the JNK/c-Jun signaling pathway upstream of Rac1 in a transcription-independent manner, demonstrating a novel mechanism of E1A action.

Chen J, Solomides C, Simpkins H
Sensitization of mesothelioma cells to platinum-based chemotherapy by GSTπ knockdown.
Biochem Biophys Res Commun. 2014; 447(1):77-82 [PubMed] Related Publications
It is predicted that the incidence of mesothelioma will increase and thus it is important to find new ways to treat this chemoresistant tumor. Glutathione-S-Transferase π (GSTπ) is found at significant levels in mesotheliomas and thus attenuating its intracellular levels may provide a means of sensitizing mesothelioma cells to chemotherapy. GSTπ knockdowns were therefore prepared with shRNA (less off-target effects) employing two cell lines (211H, H2452) that were typed by immunohistochemistry to be of mesothelial origin. The knockdowns exhibited a decrease in both total GST enzyme activity and GSTπ protein levels as well as an increase in both glutathione levels and sensitivity to cis and oxaliplatin. Cisplatin treatment of the knockdowns increased ROS levels significantly (as compared to the parental cells) and produced activation of the JNK/p38 pathways and activating transcription factor (ATF2). The degree of activation and increase in ROS appeared to correlate with the cell line's sensitivity to cisplatin. Treatment with N-Acetyl Cysteine decreased ROS production and JNK/p38 phosphorylation but had minimal affect on ATF2 suggesting a direct interaction of GTPπ with this transcription factor. Oxaliplatin treatment produced only minimal changes in ROS levels and activation of the JNK/p38 pathway. Recently, new methods of siRNA delivery (nanoparticles) have been shown to be effective in decreasing the levels of target proteins in humans including candidate genes involved in drug resistance - thus this approach may have promise in sensitizing cisplatin-resistant tumors to chemotherapy.

Shin YJ, Kumarasamy V, Camacho D, Sun D
Involvement of G-quadruplex structures in regulation of human RET gene expression by small molecules in human medullary thyroid carcinoma TT cells.
Oncogene. 2015; 34(10):1292-9 [PubMed] Related Publications
The RET (rearranged during transfection) proto-oncogene encodes a receptor tyrosine kinase for members of the glial cell line-derived neurotrophic factor family of extracellular signaling molecules. The activating germline point mutations in the RET, which are known to induce oncogenic activation of RET tyrosine kinase, are associated with the development of medullary thyroid carcinoma (MTC) and pathogenesis of multiple endocrine neoplasia type 2 (MEN2). The polypurine/polypyrimidine tract in the proximal promoter region of the human RET gene (-51 to -33 relative to transcription start site) is essential for basal transcriptional activity of this gene. This tract consists of a guanine-rich sequence containing five runs of at least three contiguous guanines separated by one or more bases, conforming to a general motif capable of forming an intramolecular G-quadruplex. Here, we show that specific G-quadruplex structures formed in the RET promoter region act to repress the transcription of this gene, and transcription of this gene can be controlled by ligand-mediated G-quadruplex stabilization. In this study, NSC194598, a derivative of indeno[1,2,3-de]quinazoline, was found to be a novel G-quadruplex interactive agent that interfered with transcriptional activation of mutated RET gene in human medullary thyroid carcinoma TT cells. This compound significantly reduced endogenous RET protein levels and increased apoptosis in these cells. Our results provide further support for the idea that G-quadruplex structures may have a critical role in transcriptional regulation of the RET gene in vivo, providing insight into a novel strategy for transcriptional repression of this gene by small molecules.

Yu ZQ, Zhang BL, Ni HB, et al.
Hyperacetylation of histone H3K9 involved in the promotion of abnormally high transcription of the gdnf gene in glioma cells.
Mol Neurobiol. 2014; 50(3):914-22 [PubMed] Related Publications
The mechanism underlying abnormally high transcription of the glial cell line-derived neurotrophic factor (GDNF) gene in glioma cells is not clear. In this study, to assess histone H3K9 acetylation levels in promoters I and II of the gdnf gene in normal human brain tissue, low- and high-grade glioma tissues, normal rat astrocytes, and rat C6 glioblastoma cells, we employed chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR), real-time PCR, and a pGL3 dual fluorescence reporter system. We also investigated the influence of treatment with curcumin, a histone acetyltransferase inhibitor, and trichostatin A (TSA), a deacetylase inhibitor, on promoter acetylation and activity and messenger RNA (mRNA) expression level of the gdnf gene in C6 cells. Compared to normal brain tissue, H3K9 acetylation in promoters I and II of the gdnf gene increased significantly in high-grade glioma tissues but not in low-grade glioma tissues. Moreover, H3K9 promoter acetylation level of the gdnf gene in C6 cells was also remarkably higher than in normal astrocytes. In C6 cells, curcumin markedly decreased promoter II acetylation and activity and GDNF mRNA expression. Conversely, all three measurements were significantly increased following TSA treatment. Our results suggest that histone H3K9 hyperacetylation in promoter II of the gdnf gene might be one of the reasons for its abnormal high transcription in glioma cells.

Golubkov VS, Strongin AY
Downstream signaling and genome-wide regulatory effects of PTK7 pseudokinase and its proteolytic fragments in cancer cells.
Cell Commun Signal. 2014; 12:15 [PubMed] Article available free on PMC after 01/09/2015 Related Publications
BACKGROUND: The full-length membrane protein tyrosine kinase 7 (PTK7) pseudokinase, an important component of the planar cell polarity and the Wnt canonical and non-canonical pathways, is a subject of step-wise proteolysis in cells and tissues. The proteolysis of PTK7 involves membrane type-matrix metalloproteinase (MT1-MMP), members of the Disintegrin Domain and Metalloproteinase (ADAM) family, and γ-secretase. This multi-step proteolysis results in the generation of the digest fragments of PTK7. These fragments may be either liberated into the extracellular milieu or retained on the plasma membrane or released into the cytoplasm and then transported into the nucleus.
RESULTS: We employed the genome-wide transcriptional and kinome array analyses to determine the role of the full-length membrane PTK7 and its proteolytic fragments in the downstream regulatory mechanisms, with an emphasis on the cell migration-related genes and proteins. Using fibrosarcoma HT1080 cells stably expressing PTK7 and its mutant and truncated species, the structure of which corresponded to the major PTK7 digest fragments, we demonstrated that the full-length membrane 1-1070 PTK7, the N-terminal 1-694 soluble ectodomain fragment, and the C-terminal 622-1070 and 726-1070 fragments differentially regulate multiple genes and signaling pathways in our highly invasive cancer cell model. Immunoblotting of the selected proteins were used to validate the results of our high throughput assays.
CONCLUSIONS: Our results suggest that PTK7 levels need to be tightly controlled to enable migration and that the anti-migratory effect of the full-length membrane PTK7 is linked to the down-regulation of multiple migration-related genes and to the activation of the Akt and c-Jun pathway. In turn, the C-terminal fragments of PTK7 act predominantly via the RAS-ERK and CREB/ATF1 pathway and through the up-regulation of cadherin-11. In general, our data correlate well with the distinct functionality of the full-length receptor tyrosine kinases and their respective intracellular domain (ICD) proteolytic fragments.

Klein P, Müller-Rischart AK, Motori E, et al.
Ret rescues mitochondrial morphology and muscle degeneration of Drosophila Pink1 mutants.
EMBO J. 2014; 33(4):341-55 [PubMed] Article available free on PMC after 01/09/2015 Related Publications
Parkinson's disease (PD)-associated Pink1 and Parkin proteins are believed to function in a common pathway controlling mitochondrial clearance and trafficking. Glial cell line-derived neurotrophic factor (GDNF) and its signaling receptor Ret are neuroprotective in toxin-based animal models of PD. However, the mechanism by which GDNF/Ret protects cells from degenerating remains unclear. We investigated whether the Drosophila homolog of Ret can rescue Pink1 and park mutant phenotypes. We report that a signaling active version of Ret (Ret(MEN₂B) rescues muscle degeneration, disintegration of mitochondria and ATP content of Pink1 mutants. Interestingly, corresponding phenotypes of park mutants were not rescued, suggesting that the phenotypes of Pink1 and park mutants have partially different origins. In human neuroblastoma cells, GDNF treatment rescues morphological defects of PINK1 knockdown, without inducing mitophagy or Parkin recruitment. GDNF also rescues bioenergetic deficits of PINK knockdown cells. Furthermore, overexpression of Ret(MEN₂B) significantly improves electron transport chain complex I function in Pink1 mutant Drosophila. These results provide a novel mechanism underlying Ret-mediated cell protection in a situation relevant for human PD.

Zhao Y, Li Y, Han J, et al.
Helicobacter pylori enhances CIP2A expression and cell proliferation via JNK2/ATF2 signaling in human gastric cancer cells.
Int J Mol Med. 2014; 33(3):703-10 [PubMed] Related Publications
Helicobacter pylori (H. pylori) infection plays an important role in the development of gastric carcinomas. Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a novel human oncoprotein that functions as an important regulator of cell growth and malignant transformation. In the present study, we aimed to investigate the potential mechanisms by which H. pylori upregulates the expression of CIP2A and the functional impact of H. pylori-induced CIP2A in gastric cancer cells. We demonstrated that infection of MKN-45 cells with H. pylori led to a marked increase in the expression of CIP2A at the mRNA and protein levels. H. pylori-induced CIP2A was associated with increased cell proliferation. In addition, H. pylori was found to activate the JNK2 pathway. Importantly, both H. pylori-induced CIP2A production and cell proliferation were partially reversed by inhibition of JNK2 signaling. Similarly, the blockade of H. pylori-induced CIP2A expression by siRNA against CIP2A also inhibited cell proliferation. Thus, H. pylori appears to stimulate the expression of CIP2A and proliferation of gastric cancer cells via JNK2 signaling. These findings suggest that H. pylori-induced upregulation of CIP2A contributes to the development and progression of gastric cancer. Further in vivo studies are warranted to explore the biological role of CIP2A and its interaction with JNK2 signaling in gastric cancer.

Antonescu CR, Dal Cin P
Promiscuous genes involved in recurrent chromosomal translocations in soft tissue tumours.
Pathology. 2014; 46(2):105-12 [PubMed] Related Publications
Soft tissue tumours represent a heterogeneous group of mesenchymal lesions and their classification continues to evolve as a result of incorporating advances in cytogenetic and molecular techniques. In the last decade, traditional diagnostic approaches were supplemented with a significant number of reliable molecular diagnostic tools, detecting tumour type specific genetic alterations. Additionally, the successful application of some of these techniques to formalin fixed, paraffin embedded tissue enabled a broader range of clinical material to be subjected to molecular analysis. However, despite all these remarkable advances, the realisation that some of the genetic abnormalities are not fully histotype specific and that certain gene aberrations can be shared among different sarcoma types, otherwise completely unrelated clinically or immunophenotypically, has introduced some drawbacks in surgical pathology practice. One such common example is the presence of EWSR1 gene rearrangements by fluorescence in situ hybridisation (FISH), a test now preferred over the elaborate RT-PCR testing, in a variety of benign and highly malignant soft tissue tumours, in addition to a subset of carcinomas. Furthermore, the presence of identical gene fusions in completely different sarcoma types (i.e., EWSR1-ATF1, EWSR1-CREB1) or in non-mesenchymal malignancies (epithelial or haematological) has raised skepticism as to their diagnostic utility, and their lack of specificity has been compared to the limitations of other ancillary techniques, in particular immunohistochemistry. This review catalogues the main groups of genes that behave in a promiscuous manner within recurrent fusion events in soft tissue tumours. Although we acknowledge that the present molecular classification of soft tissue tumours is much more complex than two decades ago, when EWSR1 gene rearrangements had been described as the hallmark of Ewing sarcoma, we make the strong argument that with very few exceptions, the prevalence of fusion transcripts in most sarcomas is such that they come to define these entities and can be used as highly specific molecular diagnostic markers in the right clinical and pathological context.

Jiang S, Willox B, Zhou H, et al.
Epstein-Barr virus nuclear antigen 3C binds to BATF/IRF4 or SPI1/IRF4 composite sites and recruits Sin3A to repress CDKN2A.
Proc Natl Acad Sci U S A. 2014; 111(1):421-6 [PubMed] Article available free on PMC after 01/09/2015 Related Publications
Epstein-Barr virus nuclear antigen 3C (EBNA3C) repression of CDKN2A p14(ARF) and p16(INK4A) is essential for immortal human B-lymphoblastoid cell line (LCL) growth. EBNA3C ChIP-sequencing identified >13,000 EBNA3C sites in LCL DNA. Most EBNA3C sites were associated with active transcription; 64% were strong H3K4me1- and H3K27ac-marked enhancers and 16% were active promoters marked by H3K4me3 and H3K9ac. Using ENCODE LCL transcription factor ChIP-sequencing data, EBNA3C sites coincided (±250 bp) with RUNX3 (64%), BATF (55%), ATF2 (51%), IRF4 (41%), MEF2A (35%), PAX5 (34%), SPI1 (29%), BCL11a (28%), SP1 (26%), TCF12 (23%), NF-κB (23%), POU2F2 (23%), and RBPJ (16%). EBNA3C sites separated into five distinct clusters: (i) Sin3A, (ii) EBNA2/RBPJ, (iii) SPI1, and (iv) strong or (v) weak BATF/IRF4. EBNA3C signals were positively affected by RUNX3, BATF/IRF4 (AICE) and SPI1/IRF4 (EICE) cooccupancy. Gene set enrichment analyses correlated EBNA3C/Sin3A promoter sites with transcription down-regulation (P < 1.6 × 10(-4)). EBNA3C signals were strongest at BATF/IRF4 and SPI1/IRF4 composite sites. EBNA3C bound strongly to the p14(ARF) promoter through SPI1/IRF4/BATF/RUNX3, establishing RBPJ-, Sin3A-, and REST-mediated repression. EBNA3C immune precipitated with Sin3A and conditional EBNA3C inactivation significantly decreased Sin3A binding at the p14(ARF) promoter (P < 0.05). These data support a model in which EBNA3C binds strongly to BATF/IRF4/SPI1/RUNX3 sites to enhance transcription and recruits RBPJ/Sin3A- and REST/NRSF-repressive complexes to repress p14(ARF) and p16(INK4A) expression.

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