Loss Of Egr-1 Research Articles

Sex linked behavioral and hippocampal transcriptomic changes in mice with cell-type specific Egr1 loss

The transcription factor EGR1 is instrumental in numerous neurological processes, encompassing learning and memory as well as the reaction to stress. Egr1 complete knockout mice demonstrate decreased depressive or anxiety-like behavior and impaired performance in spatial learning and memory. Nevertheless, the specific functions of Egr1 in distinct cell types have been largely underexplored. In this study, we cataloged the behavioral and transcriptomic character of Nestin-Cre mediated Egr1 conditional knockout (Egr1cKO) mice together with their controls. Although the conditional knockout did not change nociceptive or anxiety responses, it triggered changes in female exploratory activity during anxiety testing. Hippocampus-dependent spatial learning in the object location task was unaffected, but female Egr1cKO mice did exhibit poorer retention during testing on a contextual fear conditioning task compared to males. RNA-seq data analyses revealed that the presence of the floxed Egr1 cassette or Nestin-Cre driver alone exerts a subtle influence on hippocampal gene expression. The sex-related differences were amplified in Nestin-Cre mediated Egr1 conditional knockout mice and female mice are more sensitive to the loss of Egr1 gene. Differentially expressed genes resulted from the loss of Egr1 in neuronal cell lineage were significantly associated with the regulation of Wnt signaling pathway, extracellular matrix, and axon guidance. Altogether, our results demonstrate that Nestin-Cre and the loss of Egr1 in neuronal cell lineage have distinct impacts on hippocampal gene expression in a sex-specific manner.

Sex-Linked Growth Disorder and Aberrant Pituitary Gene Expression in Nestin-Cre-Mediated Egr1 Conditional Knockout Mice.

Genes that regulate hormone release are essential for maintaining metabolism and energy balance. Egr1 encodes a transcription factor that regulates hormone production and release, and a decreased in growth hormones has been reported in Egr1 knockout mice. A reduction in growth hormones has also been observed in Nestin-Cre mice, a model frequently used to study the nervous system. Currently, it is unknown how Egr1 loss or the Nestin-Cre driver disrupt pituitary gene expression. Here, we compared the growth curves and pituitary gene expression profiles of Nestin-Cre-mediated Egr1 conditional knockout (Egr1cKO) mice with those of their controls. Reduced body weight was observed in both the Nestin-Cre and Egr1cKO mice, and the loss of Egr1 had a slightly more severe impact on female mice than on male mice. RNA-seq data analyses revealed that the sex-related differences were amplified in the Nestin-Cre-mediated Egr1 conditional knockout mice. Additionally, in the male mice, the influence of Egr1cKO on pituitary gene expression may be overridden by the Nestin-Cre driver. Differentially expressed genes associated with the Nestin-Cre driver were significantly enriched for genes related to growth factor activity and binding. Altogether, our results demonstrate that Nestin-Cre and the loss of Egr1 in the neuronal cell lineage have distinct impacts on pituitary gene expression in a sex-specific manner.

Estrogen induces EGR1 to fine-tune its actions on uterine epithelium by controlling PR signaling for successful embryo implantation.

The harmonized actions of ovarian E2 and progesterone (P4) regulate the proliferation and differentiation of uterine cells in a spatiotemporal manner. Imbalances between these hormones often lead to infertility and gynecologic diseases. Whereas numerous factors that are involved in P4 signaling have been identified, few local factors that mediate E2 actions in the uterus have been revealed. Here, we demonstrate that estrogen induces the transcription factor, early growth response 1 ( Egr1), to fine-tune its actions in uterine epithelial cells (ECs) that are responsible for uterine receptivity for embryo implantation. In the presence of exogenous gonadotrophins, ovulation, fertilization, and embryonic development normally occur in Egr1-/- mice, but these animals experience the complete failure of embryo implantation with reduced artificial decidualization. Although serum levels of E2 and P4 were comparable between Egr1+/+ and Egr1-/- mice on d 4 of pregnancy, aberrantly reduced levels of progesterone receptor in Egr1-/- uterine ECs caused enhanced E2 activity and impaired P4 response. Ultrastructural analyses revealed that Egr1-/- ECs are not fully able to provide proper uterine receptivity. Uterine mRNA landscapes in Egr1-/- mice revealed that EGR1 controls the expression of a subset of E2-regulated genes. In addition, P4 signaling was unable to modulate estrogen actions, including those that are involved in cell-cycle progression, in ECs that were deficient in EGR1. Furthermore, primary coculture of Egr1-/- ECs with Egr1+/+ stromal cells, and vice versa, supported the notion that Egr1 is required to modulate E2 actions on ECs to prepare the uterine environment for embryo implantation. In contrast to its role in ECs, loss of Egr1 in stroma significantly reduced stromal cell proliferation. Collectively, our results demonstrate that E2 induces EGR1 to streamline its actions for the preparation of uterine receptivity for embryo implantation in mice.-Kim, H.-R., Kim, Y. S., Yoon, J. A., Yang, S. C., Park, M., Seol, D.-W., Lyu, S. W., Jun, J. H., Lim, H. J., Lee, D. R., Song, H. Estrogen induces EGR1 to fine-tune its actions on uterine epithelium by controlling PR signaling for successful embryo implantation.

Egr1 deficiency induces browning of inguinal subcutaneous white adipose tissue in mice

Beige adipocyte differentiation within white adipose tissue, referred to as browning, is seen as a possible mechanism for increasing energy expenditure. The molecular regulation underlying the thermogenic browning process has not been entirely elucidated. Here, we identify the zinc finger transcription factor EGR1 as a negative regulator of the beige fat program. Loss of Egr1 in mice promotes browning in the absence of external stimulation and leads to an increase of Ucp1 expression, which encodes the key thermogenic mitochondrial uncoupling protein-1. Moreover, EGR1 is recruited to the proximal region of the Ucp1 promoter in subcutaneous inguinal white adipose tissue. Transcriptomic analysis of subcutaneous inguinal white adipose tissue in the absence of Egr1 identifies the molecular signature of white adipocyte browning downstream of Egr1 deletion and highlights a concomitant increase of beige differentiation marker and a decrease in extracellular matrix gene expression. Conversely, Egr1 overexpression in mesenchymal stem cells decreases beige adipocyte differentiation, while increasing extracellular matrix production. These results reveal a role for Egr1 in blocking energy expenditure via direct Ucp1 transcription repression and highlight Egr1 as a therapeutic target for counteracting obesity.

Mice Lacking EGR1 Have Impaired Clock Gene (BMAL1) Oscillation, Locomotor Activity, and Body Temperature.

Early growth response transcription factor 1 (EGR1) is expressed in the suprachiasmatic nucleus (SCN) after light stimulation. We used EGR1-deficient mice to address the role of EGR1 in the clock function and light-induced resetting of the clock. The diurnal rhythms of expression of the clock genes BMAL1 and PER1 in the SCN were evaluated by semi-quantitative in situ hybridization. We found no difference in the expression of PER1 mRNA between wildtype and EGR1-deficient mice; however, the daily rhythm of BMAL1 mRNA was completely abolished in the EGR1-deficient mice. In addition, we evaluated the circadian running wheel activity, telemetric locomotor activity, and core body temperature of the mice. Loss of EGR1 neither altered light-induced phase shifts at subjective night nor affected negative masking. Overall, circadian light entrainment was found in EGR1-deficient mice but they displayed a reduced locomotor activity and an altered temperature regulation compared to wild type mice. When placed in running wheels, a subpopulation of EGR1-deficient mice displayed a more disrupted activity rhythm with no measurable endogenous period length (tau). In conclusion, the present study provides the first evidence that the circadian clock in the SCN is disturbed in mice deficient of EGR1.

Egr-1, a Stress Response Transcription Factor and Myeloid Differentiation Primary Response Gene, Behaves As Tumor Suppressor in CML

The transcription factor early growth response 1 (Egr-1) gene was identified as a macrophage differentiation primary response gene, shown to be essential for and to restrict differentiation along the macrophage lineage. There’s evidence consistent with Egr-1 behaving as a tumor suppressor of leukemia, both in vivo and in vitro, including (1) loss of Egr-1 associated with therapy derived MDS and AML; (2) deregulated Egr-1 overriding blocks in myeloid differentiation, and (3) haplo-insufficiency of Egr-1 in mice leading to increased development of myeloid disorders following treatment with the potent DNA alkylating agent, N- ethyl-nitrosourea (ENU). BCR-ABL driven leukemia (Chronic Myelogenous Leukemia [CML]) was chosen as a model system to investigate the role of Egr-1 as a tumor suppressor for leukemia. CML is a disease resulting from the neoplastic transformation of hematopoietic stem cells (HSC) with the BCR-ABL oncogene. To assess the effect of Egr-1 on BCR-ABL driven leukemia, lethally irradiated syngeneic wild type mice were reconstituted with bone marrow (BM) from either wild type or Egr-1 null mice transduced with a 210-kD BCR-ABL-expressing MSCV-GFP retrovirus (bone marrow transplantation {BMT}). It was observed that loss of Egr-1 accelerated the development of BCR-ABL driven leukemia in recipient mice. Analysis of hematopoietic organs is consistent with loss of Egr-1 accelerating development of CML but not altering the nature of the disease. Cells expressing BCR-ABL that are null for Egr-1 exhibit increased survival and proliferation, which can account for some of the above observations. Substantial evidence is available that loss of Egr1 increases the leukemia initiating cell population, which can account for the more rapid development of disease. An increased population of lineage negative BM cells was observed in Egr-1-/- BCR-ABL recipient mice compared to animals transplanted with WT BCR-ABL BM. Serial BMT has shown that Egr-1-/- BCR-ABL BM has an increased leukemic burden compared to the WT counterpart. Furthermore, data from serial colony transfer experiments confirmed that loss of Egr-1 increases leukemia initiating cells. Preliminary studies on the effect of gain of Egr-1 function are consistent with the tumor suppressor behavior of Egr-1. These data as well as analysis of human CML samples will be presented. Further investigation could result in novel targets for diagnosis, prognosis, and targeted therapeutics, including strategies for activating Egr-1 expression that can be used to treat CML, as well as other leukemic diseases. Disclosures No relevant conflicts of interest to declare.

Influence of Egr-1 in cardiac tissue-derived mesenchymal stem cells in response to glucose variations.

Mesenchymal stem cells (MSCs) represent a promising cell population for cell therapy and regenerative medicine applications. However, how variations in glucose are perceived by MSC pool is still unclear. Since, glucose metabolism is cell type and tissue dependent, this must be considered when MSCs are derived from alternative sources such as the heart. The zinc finger transcription factor Egr-1 is an important early response gene, likely to play a key role in the glucose-induced response. Our aim was to investigate how short-term changes in in vitro glucose concentrations affect multipotent cardiac tissue-derived MSCs (cMSCs) in a mouse model of Egr-1 KO (Egr-1−/−). Results showed that loss of Egr-1 does not significantly influence cMSC proliferation. In contrast, responses to glucose variations were observed in wt but not in Egr-1−/− cMSCs by clonogenic assay. Phenotype analysis by RT-PCR showed that cMSCs Egr-1−/− lost the ability to regulate the glucose transporters GLUT-1 and GLUT-4 and, as expected, the Egr-1 target genes VEGF, TGFβ-1, and p300. Acetylated protein levels of H3 histone were impaired in Egr-1−/− compared to wt cMSCs. We propose that Egr-1 acts as immediate glucose biological sensor in cMSCs after a short period of stimuli, likely inducing epigenetic modifications.

Retroviral Insertional Mutagenesis In Egr1+/- mice, Haploinsufficient For a Human Del(5q) Myeloid Leukemia Gene, Develop Myeloid Neoplasms With Proviral Insertions In Genes Syntenic To Human 5q

Therapy-related myeloid neoplasm (t-MN) is a distinctive clinical syndrome occurring after treatment with chemotherapy and/or radiotherapy, typically for a primary malignant disease. Loss of the long arm of chromosome 5, del(5q), is the most common recurring cytogenetic abnormality and is observed in 40% of t-MN patients, as well as 10-15% of patients with primary MDS or AML de novo. These deletions typically encompass over 70Mb [spanning 5q14-q33] and numerous genes, making the identification of relevant del(5q) genes very challenging. In previous studies, we identified a commonly deleted region within 5q and identified Egr1 as a del(5q) haploinsufficient myeloid leukemia gene. Using Egr1+/- mice, we previously showed that Egr1 cooperates with mutations, induced by the alkylating agent, ENU, to induce a myeloproliferative disorder with ineffective erythropoiesis (MPD). However, loss of Egr1 on its own was not sufficient for the development of MPD. To identify cooperating mutations in MPDs in Egr1 haploinsufficient mice, we conducted a retroviral insertional mutagenesis (RIM) screen. Egr1 WT (n=61) and Egr1+/- (n=77) neonates were injected with the MOL4070LTR retrovirus. Although the overall survival of MOL4070LTR-treated Egr1+/- and WT controls was similar, Egr1+/- mice developed MPD or AML with a shorter latency and at a higher overall frequency than WT littermate controls. Forty-six percent of WT mice developed myeloid disease versus 61% for Egr1+/- mice, with a median survival of 474 d for Egr1 WT and 389 d for Egr1+/- mice (p=0.03).We mapped the retroviral integration sites in myeloid neoplasms from 29 WT and 46 Egr1+/- mice using barcoded splinkerette PCR and Illumina high-throughput sequencing. To identify and analyze the statistically significant common insertion sites (CISs) we used the TAPDANCE software developed by A. Sarver. In total, 159 CISs were identified in WT mice and 365 CISs were identified in Egr1+/- mice. Several of these CIS-associated genes, such as Sox4, Pim1 and Myb have been previously identified in other genetic screens according to the Retroviral Tagged Cancer Gene Database (RTCGD). As the main goal of this study is to identify mutations that cooperate with Egr1 haploinsufficiency, we were particularly interested in CISs that were identified exclusively or more frequently in Egr1+/- mice with myeloid neoplasms. The TAPDANCE software automatically identifies associations between phenotypes and CISs using Fisher's exact test with multiple testing correction. Using this analysis, we identified six CISs that were statistically associated with myeloid neoplasms in Egr1+/- mice, but not WT mice. The candidate cancer genes proximal to these proviral insertions included Evi1, Gfi1, Evi5, and Cd47. Analysis of transcript levels revealed elevated expression of Evi1, but not Gfi1, Evi5, or Cd47 in myeloid leukemias with proviral insertions proximal to these genes.Of interest was a CIS associated with Egr1+/- mice that mapped to a region of mouse chromosome 18 that is syntenic to human 5q31.2, proximal to the Dnajc18, Ecscr, Tmem173, Cxxc5 and Psd2 genes and adjacent to the commonly deleted region. Moreover, an analysis of co-occurring CISs revealed that this CIS co-occurred with a CIS that mapped to a region of mouse chromosome 13 that is syntenic to human 5q31.1, also deleted in t-MN patients and proximal to the Tifab, and H2afy genes. Of the genes in these two CISs, CXXC5, TMEM173, TIFAB, and H2AFY each show significantly decreased expression in bone marrow cells from t-MN del(5q) patients, consistent with haploinsufficiency. Identification of the relevant del(5q) genes in t-MN continues to be a challenge for developing therapeutic targets. Loss of expression of the tumor suppressor gene, EGR1, which is expressed at haploinsufficient levels in t-MN patients with a del(5q), on its own is not sufficient for the development of myeloid leukemia. Here we performed a forward genetic screen with Egr1+/- mice and have identified several candidate del(5q) genes, including CXXC5, TMEM173, TIFAB, and H2AFY, that should now be evaluated as candidate genes that cooperate with EGR1 haploinsufficiency in the pathogenesis of t-MN. The identification of aberrant pathways resulting from haploinsufficiency of EGR1 in cooperation with these del(5q) genes may potentially lead to the new therapeutic targets for t-MNs with chromosome 5 abnormalities. Disclosures:Largaespada:Discovery Genomics, Inc: Consultancy, Share Holder Other; NeoClone Biotechnology, Inc: Consultancy, Share Holder, Share Holder Other.

Stress Response Transcription Factor Egr-1 As Tumor Suppressor Of CML

The transcription factor early growth response 1 (Egr-1) gene was identified as a macrophage differentiation primary response gene, shown to be essential for and to restrict differentiation along the macrophage lineage. There’s evidence consistent with Egr-1 behaving as a tumor suppressor of leukemia, both in vivo and in vitro, including (1) loss of Egr-1 associated with treatment derived AMLs; (2) deregulated Egr-1 overriding blocks in myeloid differentiation, and (3) haplo-insufficiency of Egr-1 in mice leading to increased development of myeloid disorders following treatment with the potent DNA alkylating agent, N- ethyl-nitrosourea (ENU). BCR-ABL driven leukemia (Chronic Myelogenous Leukemia [CML]) was chosen as a model system to investigate the role of Egr-1 as a tumor suppressor for different leukemias. CML is a disease resulting from the neoplastic transformation of hematopoietic stem cells (HSC) with the BCR-ABL oncogene. The BCR-ABL protein is a constitutively active tyrosine kinase, which promotes cell survival and proliferation by means of diverse intracellular signaling pathways, thereby being the culprit for malignant transformation. Although the Tyrosine Kinase Inhibitor (TKI) imatinib mesylate (Gleevec, Novartis) is effectively used on CML patients, resistance to imatinib has been described. Thus there is a high priority to enhance our understanding of how BCR/ABL subverts normal hematopoiesis and to identify novel targets for therapy. It was observed that Egr-1 expression is reduced in bone marrow (BM) of CML patients, and its expression is further reduced in more advanced stages of CML. Consistent with this data, Egr-1 expression is reduced in BCR-ABL-expressing murine BM. The tumor suppressor role of Egr-1 in CML was validated using mouse models. Lethally irradiated syngeneic wild type mice were reconstituted with bone marrow (BM) from either wild type or Egr-1 null mice transduced with a 210-kD BCR-ABL-expressing MSCV-retrovirus (bone marrow transplantation {BMT}). Loss of Egr-1 accelerated the development of BCR-ABL driven leukemia in recipient mice. Furthermore, no statistically significant difference in the percentage of stem cells (Lin-Sca+c-Kit+, LSK) was observed between Egr-1 WT and Egr-1-/- BM. Thus, the BM stem cell compartment of the Egr-1-/- mice does not offer a quantitative advantage to justify the faster development of leukemia compared to Egr-1 WT mice. An increased population of lineage negative BM cells was observed in Egr-1-/- BCR-ABL recipients when compared to animals transplanted with WT BCR-ABL BM, consistent with more rapid development of disease. Preliminary results from serial BMT has shown that Egr-1-/- BCR-ABL BM has an increased leukemic burden when compared to the WT counterpart. Data from serial colony transfer and studies using spleens from diseased mice as well as BCR-ABL-expressing BM will be presented. These data could result in novel targets for diagnosis, prognosis, and targeted therapeutics, including strategies for activating Egr-1 expression, that can be used to treat CML, as well as other leukemic diseases. Disclosures: No relevant conflicts of interest to declare.

Egr1 and Apc Haploinsufficiency Cooperate in the Pathogenesis of Myeloid Neoplasia: A Mouse Model for Therapy-Related Myeloid Neoplasms with a Del(5q)

Abstract 117Heterozygous deletions of the long arm of chromosome 5 are among the most common abnormalities in de novo (∼15% of patients) and therapy-related myeloid neoplasms (t-MN) (∼40% of patients). Two minimally deleted segments have been identified - the minimally deleted segment within 5q31.2 is associated with de novo AML and t-MNs, whereas the other spans 5q33.1 and is associated with MDS with an isolated del(5q). Current studies support a haploinsufficiency model, in which loss of a single allele of more than one gene on 5q contributes to the development of myeloid neoplasms. Using mouse models, we previously showed that haploinsufficiency of Egr1 (5q31.2) or Apc (5q22-frequently deleted in t-MN) independently recapitulates some features of human myelodysplastic syndromes (MDS).To test the hypothesis that reduced levels of EGR1 and APC cooperate in the pathogenesis of MDS/AML, we generated mice expressing a single allele of Egr1 and Apc: Mx1-Cre+Apcfl/+Egr1+/−(Apcdel/+Egr1+/−). At 2 mos of age, we induced deletion of a single allele of Apc by injection of 3 doses of pI-pC. Survival curves clearly show that Egr1 and Apc haploinsufficiency cooperate in the development of disease with a median survival of 129 days for Apcdel/+Egr1+/− mice and 296 days for Apcdel/+mice (P<0.0001). Although disease latency was significantly shorter for Apcdel/+Egr1+/− mice, their phenotype was similar to Apcdel/+ mice, with only two exceptions. For both cohorts, mice typically developed splenomegaly and a lethal macrocytic anemia with monocytosis. Anemic mice had an increased proportion of CD71+Ter119+ erythroblasts, indicating a block in erythroid development between the early and late basophilic erythroblast stage. Two mice displayed anemia and leukocytosis (WBC 51–72 k/mL) with an increased proportion of Mac1+ cells in the spleen and Kit+ cells in the bone marrow (1 mouse). As anticipated, mice with wild type levels of Apc (Mx1-Cre-Apcfl/+) or with loss of one allele of Egr1 showed no signs of anemia.Mutations in TP53 are commonly found in t-MNs with a del(5q) and loss of Tp53 in mouse models has been shown to promote AML by enabling aberrant self renewal. To test the hypothesis that loss of TP53 may adversely advance disease development, we crossed Tp53+/− to Egr1+/− and Apcdel/+ mice. Similar to Apcdel/+Egr1+/− mice, Apcdel/+Tp53+/− mice rapidly developed macrocytic anemia with a median survival of 144 days, suggesting that partial loss of TP53 function accelerates the Apcdel/+ -induced macrocytic anemia. Triple heterozygous mice (Apcdel/+Tp53+/−Egr1+/−) had a median survival of 178 days, but survival was not statistically different than Apcdel/+Egr1+/− mice (P=0.35) suggesting that Egr1 and Tp53 loss play redundant roles in the development of disease in Apcdel/+ mice. Thus, in the context of Apc haploinsufficiency, loss of Egr1 or Tp53 function promotes erythroid failure. These results are in contrast to the setting of ribosomal protein haploinsufficiency, as is the case in MDS with an isolated del(5q), where induction of TP53 is essential for erythroid failure.It has been proposed that inactivation of TP53 (through additional TP53 mutations) would be required for progression to AML, in the setting of a 5q deletion. To this end we transduced Egr1+/−Apcdel/+ bone marrow cells with a Tp53-specific shRNA, known to reduce Tp53 transcripts by ∼90%, and transplanted them into lethally irradiated C57BL/6 mice. Although penetrance of disease was low, 2 out of 13 mice (15%) developed an aggressive AML, as compared to 0 of 12 mice transplanted with Egr1+/−Apcdel/+ cells transduced with control shRNA. These data suggest that EGR1 and APC haploinsufficiency cooperate in the development of myeloid disorders, characterized by ineffective erythropoiesis, and that further mutations, such as that achieved by complete inactivation of TP53, are required for progression to AML. Disclosures:No relevant conflicts of interest to declare.

Expression of selected gene for acquired drug resistance to EGFR-TKI in lung adenocarcinoma

BackgroundIndividualized treatment is an attractive challenge that may allow for more effective and safer treatment of human disease. Activating mutations in the epidermal growth factor receptor (EGFR) gene in lung adenocarcinoma are associated with a dramatic clinical response to EGFR-tyrosine kinase inhibitors (TKIs). However, patients often experience a relapse after treatment with EGFR-TKIs, even when the tumors are initially highly sensitive. However, the “whole picture” regarding acquired resistance remains unclear. MethodsTumor specimens were collected from 11 lung adenocarcinoma patients before and after treatment with gefitinib. The status of the EGFR and K-ras genes were investigated by PCR-based analyses. Immunohistochemistry and real-time PCR assays were used to evaluate the MET gene in terms of its tyrosine phosphorylation and amplification, respectively. The expression of HGF, PTEN, and EGR-1, and changes in the epithelial–mesenchymal transition (EMT) status including the expression of E-cadherin and gamma-catenin as epithelial markers, and vimentin and fibronectin as mesenchymal markers, were evaluated by immunohistochemistry. ResultsSeven (64%) of the gefitinib refractory tumors exhibited a secondary threonine-to-methionine mutation at codon 790 in EGFR (T790M). All of the tumors had wild type K-ras gene expression. No MET amplification was detected in any of the samples, nor was there phosphorylation of MET detected in any of the resistant samples. Neither MET gene amplification, nor the overexpression of HGF was observed in samples without the T790M mutation. A strong expression of HGF was detected in 6 of 8 specimens with the T790M mutation. Three (38%) of 8 cases showed a loss of PTEN in samples with the T790M mutation. A loss of EGR-1 was detected in 2 (29%) of 7 cases, including one tumor without PTEN. Four (57%) of 7 cases showed positive expression of phosphorylated Akt (p-Akt). A change in the EMT status between pre-and post-treatment was observed in 4 (44%) of 9 cases. In all examined samples cases, some alterations of gene or proteins were observed. ConclusionsThe current results showed that these alterations in gene or protein expression can account for all resistant mechanisms. This phenomenon suggests the existence of complicated relationships among acquired resistance-related genes.

Epidermal-growth-factor-induced proliferation of astrocytes requires Egr transcription factors

Stimulation of astrocytes with epidermal growth factor (EGF) induced proliferation and triggered the biosynthesis of the transcription factor Egr-1, involving the activation of the extracellular signal-regulated protein kinase (ERK) signaling pathway. No differences in the proliferation rate of astrocytes prepared from wild-type or Egr-1-deficient mice were detected. However, expression of a dominant-negative mutant of Egr-1 that interfered with DNA-binding of all Egr proteins prevented EGF-induced proliferation of astrocytes. Site-directed mutagenesis of two crucial cysteine residues within the zinc finger DNA-binding domain revealed that DNA-binding of the Egr-1 mutant was essential to inhibit proliferation of EGF-stimulated astrocytes. Expression of NAB2 (a negative co-regulator of Egr-1, Egr-2 and Egr-3) or a dominant-negative mutant of Elk-1 (a key regulator of Egr-1 biosynthesis) abolished EGF-induced proliferation of astrocytes. Chromatin immunoprecipitation experiments showed that Egr-1, Egr-2 and Egr-3 bound to the gene expressing basic fibroblast growth factor (bFGF) in EGF-stimulated astrocytes. Egr-2 and Egr-3 also interacted with the bFGF gene in EGF-stimulated astrocytes prepared from Egr-1-deficient mice, indicating that loss of Egr-1 is compensated by other Egr proteins. Together, these data show that Egr transcription factors are essential for conversion of the mitogenic signal of EGF into a proliferative response.

Cryptic deletion of EGR1 in association with a novel balanced t(5;22)(q31;q11.2) in a patient with myelodysplastic syndrome

Loss of chromosome 5 or deletion of 5q is commonly seen in patients with myelodysplastic syndrome (MDS). Loss of EGR1, located on 5q31, has been postulated as contributing to the pathology of MDS in patients with –5/del(5q). We report on the first case of a patient with a novel apparent balanced translocation between chromosomes 5 and 22 at breakpoints 5q31 and 22q11.2. Fluorescence in situ hybridization using a probe of EGR1 detected a cryptic deletion masked by the translocation.

Efficacy of Conventional Cytogenetics and FISH for EGR1 To Detect Deletion 5q in Hematological Disorders and To Assess Response to Treatment with Lenalidomide.

Background: In 2006 Lenalidomide received FDA approval for myelodysplasia (MDS) with interstitial deletion 5q [del(5q)] based on conventional cytogenetic (CC) results before and after treatment. In clinical practice, FISH studies of interphase nuclei with probes for EGR1 are widely used to detect del(5q), but it is not known whether this method has similar efficacy as CC. Thus, we compared CC and FISH results for various forms of del(5q).Methods: We selected 145 patients (pts) in our cytogenetic database with del(5q). These pts had various forms of del(5q) and were representative of our clinical practice. This series included 9 pts with MDS that were studied before and after treatment with Lenalidomide. CC studies were done using standard procedures to analyze 20 metaphases from bone marrow. FISH studies were done on the same specimens using stored left-over cells and standard procedures for EGR1 (5q31.2) and D5S23/D5S721 (5p15.2). The cutoff for del(5q) for FISH was <6.0% based on 200 nuclei.Results: Among 145 pts, 104 were referred for MDS, 9 acute myeloid leukemia, 9 lymphoproliferative disorders, 7 myeloproliferative disorders and 16 without clinical information. A total of 116 pts had breakpoints at del(5)(q13q33), 17 del(5)(q15q33), 8 del(5)(q11.2~q31q31~q35), 3 del(5)(q22q33) and 1 del(5)(q13q22). Del(5q) was detected in 144 pts by both CC and FISH. One pt had del(5)(q13q22) in 20/20 metaphases but was normal by FISH. Percent abnormal cells by FISH was lower in 135/145 pts compared to CC (mean difference 22.5 ± 19.6, range −42 to 98). For 9 pts with MDS, 29 specimens were collected before and after treatment with Lenalidomide: 7 achieved complete cytogenetic remission (four relapsed) and 2 showed a decrease in percent abnormal metaphases. Results of CC and FISH for these 9 pts were concordant for detection of del(5q) and trend of tumor burden due to treatment for 28/29 specimens. The discordant specimen was collected 222 days after treatment; results of CC were normal but FISH was abnormal in 19% of nuclei. Among the 29 specimens, percent abnormal cells by FISH was lower than CC in 27 (mean difference 13.9% ± 22.5, range −19.0 to 73.5). Each of the 9 treated pts had only del(5q) prior to treatment, and was detected by both FISH and CC. After treatment, FISH did not detect a separate cytogenetic clone involving deletion 20q without del(5q) in 2 pts nor did it identify development of other anomalies in 3 pts.Conclusions: Results of CC and FISH were mostly concordant, but differed in 4 respects. First, 1/145 pts was normal by FISH but had del(5q) by CC. This pt had unusual breakpoints that did not result in loss of EGR1. Second, percentage of cells with del(5q) was usually lower by FISH than CC. This observation is likely related to a different mitotic rate of cells with del(5q) versus normal cells. Third, 1 specimen was abnormal by FISH but normal by CC after treatment, but 2 subsequent specimens were concordant. This discrepancy was likely due to interference of mitosis of del(5q) cells and residual non-proliferating disease. Fourth, 2 treated pts developed a separate cytogenetic clone without del(5q) and 3 developed other anomalies that could not be detected by FISH. We believe FISH for EGR1 is useful in clinical practice, but not a substitute for CC.

Antisense to the early growth response-1 gene (Egr-1) inhibits prostate tumor development in TRAMP mice.

Egr-1 is a transcription factor induced by stress or injury, mitogens, and differentiation factors. Egr-1 regulates the expression of genes involved in growth control or survival. Expression of Egr-1 results in either promotion or regression of cell proliferation, depending on cell type and environment. Egr-1 acts as a tumor suppressor in many cell types and loss of Egr-1 has been proposed to contribute to cancer progression. There is strong new evidence however suggesting that Egr-1 overexpression is involved in prostate cancer progression. For example, Egr-1 expression levels are elevated in human prostate carcinomas in proportion to grade and stage. Furthermore, prostate cancer progression was significantly delayed in two models of prostate cancer mice lacking Egr-1. Our objective in the present study is to test whether inhibition of Egr-1 function would block cell proliferation and inhibit the transformed phenotype of prostate cancer cells in vitro and in vivo. We describe the development of high affinity and high specificity antisense oligonucleotides that efficiently inhibit Egr-1 expression. We show that inhibition of Egr-1 expression in mouse or human prostate cancer cells decreased proliferation and reduced the capacity of these cells to form colonies and to grow in soft agar. Conversely, stable expression of Egr-1 in normal human prostate epithelial 267B1 cells promoted transformation. In TRAMP mice, treatment with Egr-1 antisense oligonucleotides delayed the occurrence of prostate tumors. Importantly, Egr-1 antisense showed little or no toxicity when injected into animals. Finally, we identified a few genes such as cyclin D2, p19ink4d, and Fas that are directly regulated by Egr-1 in prostate cancer cells and that control cell cycle and survival.