PTEN regulation by Akt-EGR1-ARF-PTEN axis.

The PTEN tumour suppressor gene is induced by the early growth response 1 (EGR1) transcription factor, which also transactivates p53, p73, and p300/CBP as well as other proapoptotic and anti-cancer genes. Here, we describe a novel Akt-EGR1-alternate reading frame (ARF)-PTEN axis, in which PTEN activation in vivo requires p14ARF-mediated sumoylation of EGR1. This modification is dependent on the phosphorylation of EGR1 at S350 and T309 by Akt, which promotes interaction of EGR1 with ARF at K272 in its repressor domain by the ARF/Ubc9/SUMO system.

Egr1 regulates the coordinated expression of numerous EGF receptor target genes as identified by ChIP-on-chip.

Background: UV irradiation activates the epidermal growth factor receptor, induces Egr1 expression and promotes apoptosis in a variety of cell types. We examined the hypothesis that Egr1 regulates genes that mediate this process by use of a chip-on-chip protocol in human tumorigenic prostate M12 cells.

Results: UV irradiation led to significant binding of 288 gene promoters by Egr1.

Role of vinculin in regulating focal adhesion turnover.

Although vinculin (-/-) mouse embryo fibroblasts assemble focal adhesions (FAs), they spread more slowly, less extensively, and close a wound more rapidly than vinculin (+/+) cells. To investigate the structure and dynamics of FAs in these cells, we used real-time interference reflection microscopy (IRM) thus avoiding the need to express exogenous GFP-tagged FA proteins which may be misregulated. This showed that the FAs were smaller, less abundant and turned over more rapidly in vinculin null compared to wild-type cells.

"Promoter array" studies identify cohorts of genes directly regulated by methylation, copy number change, or transcription factor binding in human cancer cells.

DNA microarrays of promoter sequences have been developed in order to identify the profile of genes bound and activated by DNA regulatory proteins such as the transcription factors c-Jun and ATF2 as well as DNA-modifying methylases. The arrays contain 3083 unique human promoter sequences from +500 to -1000 nts from the transcription start site. Cisplatin-induced DNA damage rapidly leads to specific activation of the Jun kinase pathway leading to increased phosphorylation of c-Jun and ATF2-DNA complexes at hundreds of sites within 3 hours.

Survey of differentially methylated promoters in prostate cancer cell lines.

DNA methylation and copy number in the genomes of three immortalized prostate epithelial and five cancer cell lines (LNCaP, PC3, PC3M, PC3M-Pro4, and PC3M-LN4) were compared using a microarray-based technique. Genomic DNA is cut with a methylation-sensitive enzyme HpaII, followed by linker ligation, polymerase chain reaction (PCR) amplification, labeling, and hybridization to an array of promoter sequences. Only those parts of the genomic DNA that have unmethylated restriction sites within a few hundred base pairs generate PCR products detectable on an array.

Early growth response 1 acts as a tumor suppressor in vivo and in vitro via regulation of p53.

The early growth response 1 (Egr1) gene is a transcription factor that acts as both a tumor suppressor and a tumor promoter. Egr1-null mouse embryo fibroblasts bypass replicative senescence and exhibit a loss of DNA damage response and an apparent immortal growth, suggesting loss of p53 functions. Stringent expression analysis revealed 266 transcripts with >2-fold differential expression in Egr1-null mouse embryo fibroblasts, including 143 known genes.

Cross-regulation between Egr-1 and APE/Ref-1 during early response to oxidative stress in the human osteoblastic HOBIT cell line: evidence for an autoregulatory loop.

The Early Growth Response protein (Egr-1) is a C(2)H(2)-zinc finger-containing transcriptional regulator involved in the control of cell proliferation and apoptosis. Its DNA-binding activity is redox regulated in vitro through the oxidation-reduction of Cys residues within its DNA-binding domain. APE/Ref-1 is a DNA-repair enzyme with redox modulating activities on several transcription factors.

Co-factors p300 and CBP catch Egr1 in their network.

p300 and CBP are large (300 kDa) nuclear scaffold proteins that act as co-activators of transcription in most cell types and are over-expressed in prostate cancer. Recently, the Egr1 transcription factor was shown to up- or down-regulate p300 and CBP transcription based on the nature of its post-translational modification. Notably, interactions of the three proteins provide fine tuning for Egr1-induced growth or cell death responses.

Vinculin controls PTEN protein level by maintaining the interaction of the adherens junction protein beta-catenin with the scaffolding protein MAGI-2.

PTEN is a frequently mutated tumor suppressor in malignancies. Interestingly, some malignancies exhibit undetectable PTEN protein without mutations or loss of PTEN mRNA. The cause(s) for this reduction in PTEN is unknown.

Identification of promoters bound by c-Jun/ATF2 during rapid large-scale gene activation following genotoxic stress.

The NH2-terminal Jun kinases (JNKs) function in diverse roles through phosphorylation and activation of AP-1 components including ATF2 and c-Jun. However, the genes that mediate these processes are poorly understood. A model phenotype characterized by rapid activation of Jun kinase and enhanced DNA repair following cisplatin treatment was examined using chromatin immunoprecipitation with antibodies against ATF2 and c-Jun or their phosphorylated forms and hybridization to promoter arrays.

Heterozygous inactivation of the vinculin gene predisposes to stress-induced cardiomyopathy.

Vinculin and its muscle splice variant metavinculin link focal adhesions and cell-to-cell contact sites to the actin cytoskeleton. We hypothesized that normal expression of vinculin isoforms would be essential for integrity of cardiomyocytes and preservation of normal cardiac function. We studied heterozygous vinculin knockout mice (Vin+/-) that develop and breed normally.

Control of phosphatase and tensin homolog (PTEN) gene expression in normal and neoplastic thyroid cells.

The lipid phosphatase, phosphatase and tensin homolog (PTEN), is a key element in controlling cell growth and survival and has a well established role as tumor suppressor protein in many neoplasia. Several data indicate that silencing of PTEN gene expression may be relevant in follicular thyroid cell transformation. Thus, in the present study regulation of PTEN gene expression in thyroid cells was investigated.

Coactivating factors p300 and CBP are transcriptionally crossregulated by Egr1 in prostate cells, leading to divergent responses.

Related coactivators p300 and CBP affect the transcriptional activities of many transcription factors (TF), producing multiple downstream effects. Here we show that immediate early response TF, Egr1, acts upstream of p300/CBP to induce or to repress transcription, depending on the stimulus. Cells induced with serum to increase endogenous Egr1 increase the transcription of p300/CBP only when Egr1 binding sites in the promoter are not mutated, causing the expression of downstream targets of Egr1 which leads to survival and growth.

Vinculin modulation of paxillin-FAK interactions regulates ERK to control survival and motility.

Cells lacking vinculin are highly metastatic and motile. The reasons for this finding have remained unclear. Both enhanced survival and motility are critical to metastasis.

Egr1 signaling in prostate cancer.

Egr1 is a multifunctional transcription factor regulating a remarkable spectrum of cellular responses from survival to apoptosis, growth to growth arrest, differentiation to transformation, senescence as well as memory and learning effects. In prostate cancer, Egr1 levels are constitutively high and closely linked to cancer development and progression. This zinc-finger protein is a short-lived, immediate early growth response gene known to be induced by a large number of extracellular stimuli such as irradiation (all wavelengths tested), hypoxia, hyperoxia, chemotherapy agents, and more.

Anterior neural plate regionalization in cripto null mutant mouse embryos in the absence of node and primitive streak.

The relation between the role of the organizer at the gastrula stage and the activity of earlier signals in the specification, maintenance, and regionalization of the developing brain anlage is still controversial. Mouse embryos homozygous for null mutation in the cripto gene die at about 9.0 days postcoitum (d.

Nodal-dependent Cripto signaling promotes cardiomyogenesis and redirects the neural fate of embryonic stem cells.

The molecular mechanisms controlling inductive events leading to the specification and terminal differentiation of cardiomyocytes are still largely unknown. We have investigated the role of Cripto, an EGF-CFC factor, in the earliest stages of cardiomyogenesis. We find that both the timing of initiation and the duration of Cripto signaling are crucial for priming differentiation of embryonic stem (ES) cells into cardiomyocytes, indicating that Cripto acts early to determine the cardiac fate.

Insulin-like growth factor-II regulates PTEN expression in the mammary gland.

The tumor suppressor PTEN is altered in many cancers, including breast cancer, but only a handful of factors are known to control its expression. PTEN plays a vital role in cell survival and proliferation by regulating Akt phosphorylation, a key component of the phosphatidylinositol 3 kinase (PI3K) pathway. Here we show that insulin-like growth factor-II (IGF-II), which signals through PI3K, regulates PTEN expression in the mammary gland.

Early growth response 1 protein, an upstream gatekeeper of the p53 tumor suppressor, controls replicative senescence.

The proliferation of most primary cells in culture is limited by replicative senescence and crisis, p53-dependent events. However, the regulation of p53 itself has not been defined. We find that deletion of the early growth response 1 (EGR1) transcription factor leads to a striking phenotype, including complete bypass of senescence and apparent immortal growth consistent with loss of a suppressor gene.

In vivo cloning and characterization of a new growth suppressor protein TOE1 as a direct target gene of Egr1.

Egr1, an immediate early transcription factor, responds to diverse stimuli and affects gene transcription to accomplish its biological effects. One important effect of Egr1 expression is to decrease the growth and tumorigenic potential of several tumor cell types. To identify important Egr1 target genes, we have adapted a methodology involving formaldehyde-induced protein-DNA cross-linking, chromatin immunoprecipitation, and multiplex PCR.

Egr1 promotes growth and survival of prostate cancer cells. Identification of novel Egr1 target genes.

In the majority of aggressive tumorigenic prostate cancer cells, the transcription factor Egr1 is overexpressed. We provide new insights of Egr1 involvement in proliferation and survival of TRAMP C2 prostate cancer cells by the identification of several new target genes controlling growth, cell cycle progression, and apoptosis such as cyclin D2, P19ink4d, and Fas. Egr1 regulation of these genes, identified by Affymetrix microarray, was confirmed by real-time PCR, immunoblot, and chromatin immunoprecipitation assays.

Negative feedback regulation of the tumor suppressor PTEN by phosphoinositide-induced serine phosphorylation.

The PTEN tumor suppressor phosphatase directly counteracts the multiple functions of phosphatidylinositol 3-kinase by removing phosphate from the D3 position of inositol phospholipids. Like many lymphomas and leukemias, the Jurkat T cell line lacks PTEN protein due to frame-shift mutations in both PTEN alleles and therefore survives in long-term cell culture. We report that PTEN reintroduced into Jurkat was highly phosphorylated on serines 380 and 385 in its C terminus, particularly the former site.

Egr1 transcription factor: multiple roles in prostate tumor cell growth and survival.

The transcription factor, Egr1, so-called because it is encoded by the immediate early growth response gene, Egr1, is rapidly induced by growth factors to transduce the proliferative signal. The induction of Egr1 by external stimuli is generally transient but appears to be sustained in some prostate tumor cell lines and tumors, suggesting that Egr1 stimulates tumor cell growth. In contradiction, in breast, lung and brain tumors, Egr1 expression is often absent or reduced and when re-expressed, results in growth suppression.

Cripto: a tumor growth factor and more.

Cripto, a growth factor with an EGF-like domain, and the first member of the EGF-CFC family of genes to be sequenced and characterized, contributes to deregulated growth of cancer cells. A role for Cripto in tumor development has been described in the human and the mouse. Members of the EGF-CFC family are found only in vertebrates: CFC proteins in zebrafish, Xenopus, chick, mouse and human have been characterized and indicate some common general functions in development.