The Transcription Factor Egr1 Is a Direct Regulator of Multiple Tumor

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REVIEW The transcription factor Egr1 is a direct regulator of multiple tumor suppressors including TGFb1, PTEN, p53, and fibronectin V Baron1, ED Adamson2, A Calogero3, G Ragona3 and D Mercola1,4,5 1Sidney Kimmel Cancer Center, San Diego, CA, USA; 2The Burnham Institute, La Jolla, CA, USA; 3The University of Rome, Rome, Italy; 4The Rebecca and John Moores Cancer Center, University of California at San Diego, La Jolla, CA, USA and 5The Department of Pathology, University of California at Irvine, Irvine, CA, USA

Recent studies are reviewed indicating that the transcription factor early growth response-1 (Egr1) is a direct regulator of multiple tumor suppressors including TGFb1, PTEN, p53, and fibronectin. The downstream pathways of these factors display multiple nodes of interaction with each other, suggesting the existence of a functional network of suppressor factors that serve to maintain normal growth regulation and resist the emergence of transformed variants. Paradoxically, Egr1 is oncogenic in prostate cancer. In the majority of these cancers, PTEN or p53 is inactive. It is suggested that these defects in the suppressor network allow for the unopposed induction of TGFb1 and fibronectin, which favor transformation and survival of prostate tumor epithelial cells, and explain the role of Egr1 in prostate cancer. Egr1 is a novel and logical target for intervention by gene therapy methods, and targeting methods are discussed. Cancer Gene Therapy (2006) 13, 115–124. doi:10.1038/sj.cgt.7700896; published online 2 September 2005 Keywords: suppressor network; anoikis; systems biology; prostate cancer; tumor progression

Introduction products. In the case of prostate cancer, however, specific defects in this network may lead to the unopposed action Egr1 or the early growth response gene product is a zinc- of Egr1 on TGFb1 that favors tumor progression. finger transcription factor of 59 000 Da that, unusually, appears to activate transcription by binding to DNA as a monomer. The role of Egr1 in cancer was last reviewed in these pages in 1998.1 The thrust of the review was the TGFb1 emerging evidence indicating that Egr1 has significant tumor suppressor properties. Two mechanisms – the Strong evidence indicates that under physiological condi- direct induction of the epithelial cell suppressor TGFb1 tions TGFb1 is growth inhibitory for cells of epithelial origin that bear an intact TGFb1 RII receptor signal and the direct induction of p53 in melanoma cells to 2,3 promote apoptosis – were emphasized. Numerous other transduction pathway. The promoter of the human genes implicated in the regulation of cancer such as TGFb1 gene contains at least two Egr1-binding sites, both of which can bind Egr1, leading to the activation of members of the PDGF and IGF families have promoter 4,5 elements consistent with regulation by Egr1, and the transcription. The significance of this interaction in evidence that Egr1 functionally regulates these genes was human tumor cells was shown by re-expression of Egr1 in summarized. Since then, a wealth of new information has human HT1080 fibrosarcoma cells, which do not normally express Egr1 but do exhibit functional TGFb1 RII extended our mechanistic knowledge of the suppressor 5 theory, especially for the role of the TGFb1, IGF-II, receptors. HT1080 cells that have been made to express PTEN, fibronectin, p53, and p73 genes, and has added the Egr1 acquire the ability to synthesize and secrete complication that Egr1 plays an important role in the precursor TGFb1 that becomes activated and acts in progression of prostate cancer. These topics are briefly an autocrine fashion to suppress growth. Chromatin immunoprecipitation of Egr1–DNA complexes has con- reviewed and we offer the hypothesis that Egr1 partici- 6 pates in the regulation of a network of suppressor gene firmed the regulation of TGFb1 by Egr1 in living cells. Thus, Egr1-induced TGFb1 is a growth suppressor in Correspondence: Dr D Mercola, Department of Pathology, HT1080 cells in spite of the apparent mesenchymal origin University of California at Irvine, Irvine, CA 92017, USA. of these cells. A number of additional studies have E-mail: [email protected] documented functional TGFb1 expression in correlation Received 2 March 2005; revised 17 June 2005; accepted 27 June with or following expression of Egr1 in other cells types as 2005; published online 2 September 2005 well as in vivo (Table 1). In addition to HT1080 cells, this Egr1 is a potential target for gene therapy of prostate cancer V Baron et al 116 Table 1 Evidence for the regulation of key suppressor and growth modulating factors by Egr-1

Suppressor factor/ System Mechanisma References growth regulator

TGFb1 Human fibrosarcoma cells, HT1080 Direct Liu et al.5; de Belle et al.6 Transgenic mouse lung tissue Indirect Lee et al.11 Vascular smooth muscle cells Indirect Fu et al.85 In vivo mouse arterial stress model Indirect Sho et al.86 Human non-small-cell lung carcinoma cells Indirect Kane et al.87 Airway smooth muscle cell preparation Indirect McKay et al.88 Monkey kidney, CV-1 cells Direct Dey et al.4

PTEN Human 293T fibroblasts Direct Virolle et al.20 Mouse embryo fibroblasts Direct Virolle et al.20 Human squamous carcinoma cells (SDCCTF) Indirect Okamura et al.25 Normal FRTL-5 rat thyroid cells vs thyroid tumor cell lines, Indirect Tell et al.24 ARO, and BCPAP Fetal kidney epithelial cells Indirect Tsugawa et al.21 Mouse mammary gland in vivo Direct Moorehead et al.22 Human prostate, DU145, and lung H226 carcinoma cells Indirect Han et al.23 Human fresh surgical NSCLC Indirect Ferraro et al.32

BCL-2 v-sis-transformed mouse NIH-3T3 cells Indirect Huang et al.89

p53 Wild-type and Egr1 (À/À) mouse embryo fibroblasts Direct Krones-Herzig et al.37; Krones-Herzig et al.36

Wild-type and Egr1 (À/À) mouse embryo fibroblasts Indirect Calogero et al.14 Fresh surgical human glioblastoma series Indirect Krones-Herzig et al.90 Wild-type and Egr1 (À/À) mouse embryo fibroblasts Indirect Han et al.91 B-cell lymphoma cells BKS-2 from CBA mice Indirect Ahmed et al.92 Human A375-C6 melanoma cells Direct Huang et al.93 Mouse NIH-3T3 cells Indirect

p73 Human genomic DNA Indirect Ding et al.39 Murine cells, Neuro-2A cells Indirect Pignatalli et al.40 Human cells, HI299 Indirect Kartecheva et al.41

Fibronectin Human fibrosarcoma cells, HT1080 Direct Liu et al.7 Human glioblastoma, U251 cells Direct Liu et al.13 Human surgical glioblastoma and primary cell lines Indirect Calogero et al.15 Human fibrosarcoma cells, HT1080 Indirect de Belle et al.45 a‘Direct’ indicates that there is experimental evidence that Egr1 binds the promoter in question at regulatory sequences to affect transcription, whereas ‘indirect’ refers to published data consistent with a regulatory role of Egr1 over the level of the factor without explicit demonstration of the mechanism of regulation.

suppression mechanism has been observed in human mens of glioblastoma14 or primary cultures of the glioblastoma cells7 and in the mouse.8 In connective tissue tumors15 commonly exhibit low or undetectable levels of cells, TGFb1-induced fibrosis in a variety of settings may Egr1 consistent with a role of Egr1 in the cause or also be attributed to Egr1.9–11 Thus, TGFb1 appears to be development of glioblastoma.13,14 Re-expression of Egr1 in a general effector of Egr1-dependent growth regulation. several human glioblastoma cell lines such as U251 led to increased expression of TGFb1, fibronectin, and PAI-1.13 Surprisingly, direct addition of activated TGFb1 to the cells induced expression of PAI-1 but not fibronectin. This Fibronectin and plasminogen activator inhibitor-1 (PAI-I) led to the discovery that fibronectin is a direct target gene of Egr1 via two closely spaced Egr1-binding sequences in Potential targets of the TGFb1/Smad signal transduction the proximal promoter.13 Both fibronectin and PAI-1 pathway that may explain the transformation-suppressor induced by Egr1/TGFb1 are secreted and become part of activity of TGFb1 include fibronectin and PAI-1.2,12 This the extracellular matrix where they greatly increase possibility was examined in several human glioblastoma attachment, an effect that is blocked by addition of cells. Established human cell lines13 and surgical speci- RGD-containing peptides or neutralizing antibodies. The

Cancer Gene Therapy Egr1 is a potential target for gene therapy of prostate cancer V Baron et al 117 results are associated with slower growth and a flattened, regulation of PTEN in a variety of human cell types and more epithelioid phenotype. Consistent with these results, in the Egr1-null mouse system (Table 1). In 293T human expression of fibronectin alone in HT1080 cells reverses fetal kidney cells, Egr1 activates a PTEN-promoter-driven key aspects of transformation,16 similar to the effects of luciferase reporter system, which is inhibited by low Egr1 expression.17 Indeed, Egr1 stimulates coordinated concentrations of Egr1 antisense oligonucleotides. expression of TGFb1, fibronectin, and PAI-1. All the EMSA, mutation analysis of DNA-binding sequence, three have been found to participate in the restoration of and chromatin immunoprecipitation of Egr1–PTEN a normal phenotype in HT1080 cells.7 Comparable promoter complexes from living cells have been used to examples have been observed by others (Table 1), test the functional role of this site. These results indicating that this mechanism may be general. indicate that Egr1 is a direct and functional regulator of Fibronectin and PAI-1 are known to contribute to the PTEN gene. anchorage-dependent growth control.18,19 One mechan- Irradiation of wild-type mice strongly induces Egr1 and ism involves the interaction of fibronectin with specific PTEN mRNA; however, PTEN expression is not induced integrins, leading to activation of the protein kinase-B/ in irradiated Egr1-null mice, indicating that Egr1 Akt (PKB/AkT) pathway19 (Figure 1). This pathway is regulates the expression of PTEN in the whole animal.20 a major site of action of the tumor suppressor PTEN, The functional significance of PTEN regulation by Egr1 is thereby suggesting a potential crosstalk downstream of suggested further by the observation that MEFs from fibronectin, as described below. Egr1-null mice are resistant to apoptosis following UV irradiation, whereas MEFs from wild-type littermates rapidly undergo apoptosis following UV irradiation. The PTEN apoptosis response could be rescued by exogenous expression of Egr1 in MEFs from the Egr1-null mice. The tumor suppressor gene, PTEN/MMAC1/TEP1,is These observations provide an additional mechanism for a proapoptotic factor that is frequently suppressed or the tumor suppressor activity of Egr1. In addition to the mouse system and human cells mutated in a variety of human cancers (reviewed in 20 Virolle et al.20). Egr1 is one of the first transcription (MEFs, HT1080, 293T ), consistent significant effects have been observed in other cells and tissues,21–25 factors shown to directly regulate the expression of PTEN 22 and the first shown to regulate PTEN in living cells.20 (Table 1). Moorehead et al. observed that Egr1 is The proximal promoter of PTEN is GC rich and contains induced by and required for IGF-II-dependent induction one functional Egr1-binding site. We have examined the of PTEN in the mouse mammary gland, an effect that is absent in Egr1-null mice. Human squamous carcinoma cells, SCCTF, can be stimulated to apoptosis by treatment with calyculin A, a potent inducer of apoptosis in several β-Catenin 25 Apoptosis cell lines. Treatment of these cells with Calyculin A NFkβ causes elevation of Egr1 expression as well as its GSK Fas signal phosphorylation, which is known to increase its trans- p27 26,27 IkB Caspase 9 activation potential. This coincides with an elevation 25 FK HR Bad of PTEN expression. RNA interference specific for the • Egr1 gene inhibited not only Egr1 expression but also PKB/Akt PIP3 PTEN expression in these cells. These observations also ECM support the view that Egr1 regulates PTEN expression PIP3 ras during the initial steps of the apoptotic pathway. Silencing PTEN PI3K FAK • • FN Int of Egr1 gene expression has also been revealing in the case Shc 24 PIP2 of rat follicular thyroid cell transformation. RT-PCR experiments performed on human thyroid tumors showed Proapoptotic Survival that the absence of Egr1 mRNA is always paralleled by Pathway Pathway the absence of PTEN mRNA, suggesting that Egr1- Egr1 dependent mechanisms may play a role in the absence of PTEN gene expression that occurs during thyroid cell 24 Figure 1 Potential interaction between two known Egr1 target transformation. genes, PTEN and fibronectin, achieves a delicate balance between Egr1 protein levels are suppressed or absent in a variety attachment and apoptosis of detached cells (anoikis). It is known that of human tumor cell lines13,28 and tumors, including Egr1 directly stimulates the expression and secretion of functional breast carcinoma,28 glioblastoma,14,15 lung cancer,29,30 fibronectin, leading to RGD-dependent attachment and FAK activa- and lymphoma.31 Moreover, in the case of human non- tion. This pathway activates Akt in other systems, thereby inhibiting small-cell lung cancer, Ferraro et al.32 examined an apoptosis and promoting survival. Cells with decreased attachment properties – a manifestation of transformation – experience extensive series of samples from over 125 patients and relatively increased PTEN-mediated inhibition of Akt and increased observed that Egr1 expression predicts both PTEN level apoptosis (anoikis). Thus, Egr1 favors attachment and survival of and survival to a high degree of significance. Low levels of normal cells but apoptosis of abnormal forms with deficient Egr1 expression are associated with poor outcome and attachment properties. may have tumors resistant to therapy secondary to loss of

Cancer Gene Therapy Egr1 is a potential target for gene therapy of prostate cancer V Baron et al 118 pathways such as PTEN. It would be interesting to mechanisms, including replicative senescence and DNA determine if low Egr1 expression is correlated with damage-induced growth arrest. Thus, regulation of p53 decreased PTEN expression in other cancer types. expression by Egr1 plays a central functional role in p53-dependent growth control in the mouse.36 Consistent results have been observed in a variety of human cell and tissue systems (Table 1). For example, p53 p53Family: p53 is mutated in the majority of fresh human glioblastoma and these tumors exhibit near normal levels of Egr1.14 Egr1 directly induces the transcription of p53 and may Conversely, in the minority of cases with wild-type p53, also bind with p53 in a cytoplasmic complex, although the 33 Egr1 mRNA and protein is significantly suppressed or significance of this interaction is not clear. The stability absent,14 suggesting that progression of human glioblas- of p53 is under extensive and complex regulation largely toma is associated with selection for either silencing Egr1 owing to intricate cytoplasmic and nuclear interactions, expression or inactivating mutations of p53, but not both. especially with Mdm2 and p14ARF family members and 34 The mechanistic basis for these findings has recently related proteins (Figure 2) (reviewed in Nair et al. and 37 35 been suggested by Krones-Herzig et al. The mouse and Liu et al. ). Thus, significant regulation of p53 expression human promoters of p53 both contain at least two by the direct induction of transcription may be unusual. sequences consistent with Egr1-binding sites. In the case Direct binding of the p53 promoter by Egr1 was first of the mouse, two sites have been shown to bind nuclear shown in human melanoma A375-C6 cells during 34 extracts of wild-type Egr1 cells but not from extracts of thapsigargin-induced apoptosis. Studies of the Egr1- Egr1-null MEFs. Antibody and oligonucleotide studies null mouse system have revealed that Egr1 normally confirm the specificity of binding of these sites by Egr1. controls p53 expression leading to cell cycle arrest, Moreover, chromatin immunoprecipitation of Egr1– senescence, and – for cells that survive ‘crisis’ – DNA complexes from living wild-type but not null MEFs transformation. Freshly prepared primary fibroblasts and human tumor cells treated with Egr1 inducers but not (MEFs) from Egr1-null embryos exhibit a surprising from untreated cells have been isolated, indicating that phenotype characterized by rapid and apparent immortal Egr1 is an in vivo regulator of p53 in the mouse and growth, with no hint of replication-dependent growth 35 human. Expression analysis of the wild-type MEFs arrest characteristic of MEFs from wild-type mice. reveals significantly increased transcript levels of p53 These cells express little or no p53 although the gene is and nine known target genes of p53 compared to Egr1- intact. Thus, the Egr1-null cells were found to be null cells. Many additional transcript increases and insensitive to genotoxic stress and did not arrest following decreases were also observed, of which 143 were known DNA damage as wild-type MEFs do. The properties of genes with the same significance level as the p53-regulated replicative senescence and DNA damage-induced growth target genes. Using a bioinformatics approach, it was arrest can be rescued by infection of the Egr1-null MEFs found that 66 of these occur in the reported literature in with a retrovirus expressing either p53 or Egr1. However, correlation with changes in Egr1. Program-assisted path- the Egr1-expressing retrovirus is ineffective at restoring way construction showed that all of these informative replicative senescence or the DNA damage response in observations could be accommodated on a single regu- p53-null MEFs, indicating that Egr1 acts upstream of p53. latory pathway consisting of only four branch points. In Expression of Egr1 in Egr1-null MEFs is indeed accom- addition to p53, TGFb1, IGF-1, and IL-6 form branch panied by increased p53 protein. These studies indicate that points leading to up- or downregulation of all informative Egr1 is a key regulator of p53. Lack of Egr1 in MEFs leads significant transcript changes of the expression analysis. to suppression of key p53-dependent growth regulation The experimental observations of the direct regulation of p53 in MEFs by Egr1, together with the consistency of the expression analysis in the available literature, supports the p21 arf· Mdm2 concept that Egr1 is a functional regulator of p53 and arf p53-regulated genes in mouse MEFs. TGFβ1 p53* p53· PMdm2 degradation

nuclear ubiquitin Egr1 P- Mdm2 p53 Family: p73 and p63

PTEN PIP3/PI3K/Akt Several of the Egr1-dependent properties of p53 are Egr1 de novo cytoplasmic Mdm2 reflected in the functions of p73, a member of the p53 family that undergoes multiple splicing effects, producing Figure 2 Interactions between the two known Egr1 target genes, multiple isoforms. The regulation of p73 at the transcrip- PTEN and p53. p53 controls the expression of PTEN and Mdm2, which in turns binds to and limits the amount of active p53. p53 tional level is controlled by two promoters that produce controls the expression of the cell cycle inhibitor p21, which also is two main forms; one is named TAp73 because it contains regulated by Egr1 through direct induction of TGFb1. Thus, Egr1, the transactivating domain (TA) that gives it activities TGFb1, and p21 work to suppress cell cycling while the Egr1/PTEN similar to those of p53. The other promoter regulates the path limits the role of p53. expression of a set of isoforms known as DNp73, which

Cancer Gene Therapy Egr1 is a potential target for gene therapy of prostate cancer V Baron et al 119 lacks the same transactivating domain and therefore that PTEN inhibits activation of Akt by dephosphoryla- generates effects distinct from those of p53. In fact, many tion of the PtdIns 3-kinase activator phospho-inositol of the roles of DNp73 are oncogenic, and are possibly triphosphate (PIP3), hence restricting Mdm2 to the related to a distinct set of target genes that can be cytoplasm in human U87 glioblastoma cells.42,43 Restric- transcriptionally regulated.38 tion of Mdm2 to the cytoplasm preserves p53 function Ding et al.39 noticed that there were putative Egr1- and promotes growth arrest and apoptosis of the cancer binding sites in the p73 promoter and later it was shown cells following DNA-damaging chemotherapy. It has that Egr1 can upregulate the p73 gene to induce the been suggested that there is a direct connection between apoptosis of neuroblastoma cells.40 Kartasheva et al.41 these two major tumor suppressors that act together to noted that DNp73 can also regulate the expression of a respond to stresses and transformation. Moreover, as variety of genes independently of p53, and that Egr1 is shown here, both p53 and PTEN are direct targets of the an important player in this effect. Our recent results in Egr1 transcription factor in a variety of cell types. this regard (unpublished data of Jianxiu Yu and ED Adamson) indicate that a complicated set of feedback loops come into play between the p53 and p73 promoters and Egr1, further underlying the importance of a network p53–PTEN interactions of stress response genes that induce growth arrest, stress response, and apoptosis. The p63 family of gene products Another example of network effects is illustrated by the observation that p53 itself is a strong transactivator of that are related to p53 may also play a role but they 44 appear not to be regulated by Egr1. The p63 and p73 the PTEN promoter. This induction is augmented by genes are infrequently mutated in cancers and this is also Egr1 (unpublished data of I de Belle and ED Adamson). true for Egr1. Therefore, TAp73 and TAp63 could act in Thus, the crosstalk between PTEN and p53 is expected to place of p53 in p53-null cancer cells when an apoptosis be favored by an increase in Egr1 and disrupted by response is required to remove damaged cells. suppression of Egr1 – a common circumstance in many human cancers. The possibility that Egr1 deficiency has effects in vivo has been shown by the analysis of Egr1-null Egr1 is a suppressor in vivo mice. In Egr1-null mice, the level of PTEN expression is reduced in all tissues except the thymus and is uninducible 20 Egr1-null mice are prone to induction of skin tumors.37 even in the thymus. This observation provides strong In these studies, wild-type and Egr1-null mice were support for the thesis that deficiencies of Egr1 expression challenged with the two-step skin carcinogenesis model in vivo may have consequences on the tumor suppressor by treatment of the skin with the mutagen DMBA (7,12- function of PTEN in vivo. dimethylbenzanthracene), followed by treatment with the tumor promoter tetraphorbol acetic acid. Wild-type mice PTEN and TGFb1/fibronectin compete for regulation developed tumors, on average, 11 weeks after induction, of apoptosis whereas mice treated with either agent alone or vehicle At least two additional Egr1 target genes, fibronectin and whether wild type, null, or heterozygous for the Egr1 gene TGFb1, display downstream signaling activities that did not develop tumors. In contrast, Egr1-null mice potentially interact with the p53–PTEN network. Fibro- exposed to the complete protocol uniformly developed nectin, by virtue of its binding and activation of specific tumors with an average time of appearance of 6 weeks – a integrins such as b1 is a major mediator of cell significant difference and a time when no tumors were attachment, activation of focal adhesion kinase (FAK), 37 apparent in the wild-type control mice. and cell survival.18 Integrin binding leads to activation of the antiapoptotic pathway Akt via activation of FAK.18 As described earlier, expression of Egr1 in HT1080 The suppressor network concept human fibrosarcoma cells results in increased secretion PTEN–Mdm2 interactions of functional fibronectin7,13 and activation of FAK.45 A major regulator of cellular levels of active p53 is the Activated Akt is a major modulator of apoptosis at formation of p53–protein complexes in the nucleus several levels (reviewed in Ruoslahti18 and Kishimoto involving the mouse double minute2 gene product et al.47) (Figure 1). It phosphorylates and inactivates the (Mdm2) or the human homolog, the p53-binding protein. proapoptotic factors BAD and Caspase 8. Akt prevents Mdm2 is an oncoprotein that leads to sequestration of the release of cytochrome c from mitochondria and active p53 and also, owing to the ligase activity of Mdm2, inactivates the forkhead transcription factors (FKLR), ubiquitination and proteosomal degradation, thereby which are known for their proapoptotic effects. More- further limiting the amount of active p53 (reviewed in over, Akt mediates integrin-induced activation of the anti- Kartasheva et al.41 and Mayo and Donner42) (Figure 2). apoptotic factor Bcl-2. Thus, activation of Akt constitutes Recent observations show that phosphoinositide 3-kinase an important cell survival mechanism. Concomitantly, the (PtdIns 3-kinase, PI3K)-Akt signaling promotes the activity of Akt is opposed by the action of PTEN. These phosphorylation and translocation of the Mdm2 onco- influences establish a critical balance (Figure 1). In the protein into the nucleus, thus facilitating the down- absence of PTEN, any alteration that gives rise to regulation of p53.42,43 Donner and co-workers observed autonomous activation of the integrin/FAK/Akt pathway

Cancer Gene Therapy Egr1 is a potential target for gene therapy of prostate cancer V Baron et al 120 may favor the survival of unattached cells. In the presence MDM2 of PTEN, the Akt survival pathway is opposed by the p53 p53 p53-MDM2 PTEN-catalyzed hydrolysis of PIP3, thereby increasing Degradation active apoptosis of unattached cells, a phenomenon termed ARF ARF/MDM2

‘anoikis’ (Gr., oikos, home; anoikos, homelessness) by Cipl/waf1 18 46 p21 Ruoslahti and Frisch and Screaton. This mechanism INK4a p16 protects normal cells from the emergence of anchorage- GROWTH INK4b p15 independent populations. Cells that gain autonomous Akt TGFβ1 ARREST CIP1 p21 activity, such as occurs upon LOH, or complete deletion or KIP1 p27 inactivating mutations of PTEN, may escape this mechan- NORMAL ism. Indeed, genetically altered mice that are heterozygous EGR1 PTEN PHENOTYPE for inactivating mutations of PTEN exhibit constitutively no apoptosis apoptosis of FAK/Akt of attached ANCHORAGE elevated Akt activity in a variety of tumors including detached cells the thyroid, endometrium, breast, and prostate carcinomas cells DEPENDENCE as well as T-cell lymphomas.47 We propose that Egr1, by FN inducing the expression of functional PTEN as well as functional fibronectin and other attachment factors such as PAI-1, contributes to the maintenance of the critical balance of survival vs apoptosis determined by Akt activity Figure 3 Summary of the overall network of suppression factors (Figure 1). that are under the direct control of Egr1: TGFb1, PTEN, p53, and fibronectin. The solid arrows from Egr1 indicate regulation of target genes by direct promoter binding known in one or more human cell types to be under the direct regulation by Egr1. The subsequent TGFb1 interacts at several levels downstream interactions have been observed in one or more of these systems except the potential roles of the Inks, Akt, and Kip1, The TGFb1 signaling pathway also interacts with the which are taken from known interactions in other systems. network. First, as noted, TGFb1-induced PAI-1 is secreted and functions to facilitate cell attachment.13 Paradoxically, increased expression of Egr1 is consis- Second, several growth inhibitors such as p27Kip1Cip148–50 tently observed in prostate cancer where growing evidence and p21Cip1/Waf151–53 are regulated by the TGFb1 signal indicates that Egr1 is oncogenic. Russell and co-workers55 transduction pathway. p21Waf1 is also under the direct observed that Egr1 mRNA levels were elevated in 12 of 12 regulation of p53, indicating a potential cooperation cases of organ-confined prostate cancer but not in breast between Egr1-induced p53 and Egr1-induced TGFb1in or ovarian cancers, or in rapidly dividing rat ventral controlling cell growth. In certain cell types such as prostate cells. Egr1 mRNA was detected in epithelial and HT1080,54 fibronectin is known to be a direct target gene stromal cells at tumor margins but not in lymph node of the TGFb1 signal transduction pathway, thereby metastases. The correlation was extended to protein cooperating with Egr1-induced fibronectin. Thus, Egr1- expression. In addition, Egr1 expression was significantly induced TGFb1 may lead to several cooperative inter- increased in tumors with Gleason scores of 8–10.56 actions with tumor suppressor networks (Figure 1). The protein NAB2 (NGF-1A-binding protein), which In summary, recent studies indicate that Egr1 is a direct represses the transcriptional activity of Egr1, is down- regulator of at least four major suppressors: TGFb1, regulated in primary prostate carcinomas.57 Thus, both PTEN, p53 family members, and fibronectin. These upregulation of Egr1 and loss of its repressor NAB2 may factors have several overlapping functions, suggesting contribute to increased levels of Egr1 activity in human that cooperative interactions occur that favor the main- prostate cancer. The functional effects were demonstrated tenance of the normal cell phenotype and work to elimi- by Milbrandt and co-workers who examined the TRAMP nate the emergence of transformed cells. The combined mouse model of prostate cancer following the crossing of direct regulatory effects of Egr1 and the potential down- these mice with Egr1-null mice.58 TRAMP mice sponta- stream effects are summarized in Figure 3. neously develop prostate cancer owing to the expression of the SV40 T-antigen under the control of a prostate- specific probasin promoter. Owing to SV40 T-antigen Egr1-induced oncogenesis of prostate cancer expression, it is likely that p53 is inactivated. Tumor initiation and tumor growth rate were not affected by the Consistent with a role as a tumor suppressor, deletion of lack of Egr1; however, Egr1 deficiency significantly the Egr1-containing 5q31 region has been associated with delayed the progression from prostatic intraepithelial other malignant conditions such as lymphoma of Bloom’s neoplasia to invasive carcinoma. Further evidence for a Syndrome31 and small cell lung carcinoma (SCLC).29 role of Egr1 in oncogenesis was provided by experiments Decreased or absent Egr1 protein expression occurs in using specific high-affinity Egr1 antisense oligonucleo- NSCLC30 and glioblastoma.14 The observations indicate tides.59–61 It was shown that in a series of mouse and that Egr1 expression is commonly suppressed in tumors as human prostate cancer cells, Egr1 antisense inhibited cell has been observed in cells lines.13,28 proliferation, colony formation, and growth in soft agar,

Cancer Gene Therapy Egr1 is a potential target for gene therapy of prostate cancer V Baron et al 121 which are hallmarks of transformed cells in vitro.61,62 has diverse roles and may have the net effect of promoting Thus, Egr1 antisense reversed the transformed phenotype the progression of prostate cancer (reviewed in Albrecht of the cancer cells. Moreover, TRAMP mice have been et al.72 and Zheng et al.73). TGFb1 may also facilitate treated systemically with antisense Egr1 or control detachment and metastasis.76 An additional important oligonucleotide by i.p. injection every other day from 10 mechanism of tumor growth promotion by TGFb1may weeks of age – a time when tumors are established – to be via autocrine stimulation of endothelin-1, implicated in 20 weeks of age. The antisense oligonucleotides reduced prostate tumor progression.77 Thus, it may be speculated Egr1 protein expression in vivo and significantly reduced that in the absence of PTEN and/or p53, Egr1 becomes an the frequency and delayed the occurrence of prostate oncogenic agent in prostate cancer via the effects of its target tumors.61,62 The observations strongly indicate a unique genes, fibronectin and TGFb1. Furthermore, the oncogenic role for Egr1 in regulating the transition from localized effect of TGFb is not restricted to prostate cancer. Recently, carcinoma in situ to invasive carcinoma. the progression and metastases of breast cancer in the The basis of the oncogenic role of Egr1 in prostate transgenic mouse was shown to be driven by autocrine cancer is not known in detail. Two studies have suggested TGFb1 secretion, especially during the stage when epithelial possible Egr1 target genes in prostate cancer cells. breast cells are transforming into mesenchymal cell types.78 Expression analysis performed on prostate cancer cells While it is of interest that the tumor suppressor LAPC4 where Egr1 overexpression was driven by network can both accommodate growth suppression and adenovirus-mediated transfection identified a number of the oncogenic role of Egr1, this hypothetical basis for an growth factors, such as IGF-II, PDGF-A, and TGFb1.63 oncogenic role of Egr1 remains untested. The preponder- Interestingly, several genes identified in this screen ance of experimental evidence, however, strongly indicates are associated with neuroendocrine differentiation, which that in formed prostate carcinomas, Egr1 is oncogenic. is known to occur frequently in murine prostate cancer. We suggest that Egr1 is a valid target for suppression in Another Affymetrix microarray analysis compared mouse prostate cancer by gene therapy methods. prostate cancer cells TRAMP-C2 in the presence or absence of Egr1 antisense.64 A total of 960 genes were regulated by endogenous Egr1 and several new targets were identified. Some of these play important roles in cell Targeting Egr1 for gene therapy of prostate cancer proliferation or apoptosis, such as cyclin D2, p19INK, Fas, and TGFb1. Direct regulation of the expression of How can one exploit the indications that Egr1 promotes these genes was confirmed by quantitative PCR, Western prostate cancer for therapy in view of the arguments that analysis, and chromatin immunoprecipitation. Egr1 is a suppressor factor in normal tissue via regulation The network concept of tumor suppressors provides of a network of well-known suppressor factors? Two additional suggestions for the basis of the oncogenic approaches are apparent. First, in the whole animal, properties of Egr1 in prostate cancer. For example, in the temporary suppression of Egr1 in nonprostatic tissue may case of prostate cancer, PTEN is the most commonly not be disastrous. For example, Egr1-null mice – unlike altered gene known. The region on chromosome 10q that PTEN- and p53-null mice – do not experience an harbors the PTEN gene is hemizygously deleted in many increased incidence of tumors. This may be related to human cancers, with a frequency reaching 60–80% in the the fact that other Egr1 family members compensate for case of prostate cancer.65–67 Interestingly, even in prostate the absence of Egr1.79–81 For example, Egr4 can cancer cells that display PTEN normally, Egr1 was compensate for the deficiency of Egr1, which causes unable to regulate PTEN expression61 (as in unpublished failure of LH receptor expression and sterility owing to results). This may be because of hypermethylation of failure of luteinizing hormone regulation and Leydig cell PTEN promoter, which prevents Egr1 from binding (our steroidogenesis. If this compensatory mechanism func- unpublished results). PTEN hypermethylation occurs in tions ‘acutely’, systemic agents that inhibit the expression about 50% of prostate cancer cases.68 Thus, in prostate of Egr1 may be appropriate at least for the relatively short tumors where PTEN is not mutated or deleted, term that therapy would be applied. Effective high- hypermethylation of its promoter may preclude regula- affinity antisense agents that block Egr1 expression in tion by Egr1. In addition, p53 is inactivated in up to the mouse are known.61,62 25–50% of prostate cancer cases.69 Therefore, PTEN Second, localized irradiation therapy may be combined and/or p53 are inactivated in the majority of prostate with radiosensitive vectors that express proapoptic agents cancers. In these cases, Egr1 may have an unopposed or Egr1 antisense or iRNA. Intriguingly, this approach effect on Akt activation by continued induction of may best utilize the Egr1 promoter itself. In a series of fibronectin. Fibronectin has long been associated with studies, Weischselbaum and co-workers have exploited the stimulation of anchorage-independent growth.70 The the multiple CG-rich so-called CarG-boxes of the Egr1 expression of fibronectin correlates with features of promoter to prepare a variety of expression vectors that aggression such as metastases in prostate cancer71,72 and are induced by irradiation.82–84 For example, the radio- is a known activator of the Akt pathway in prostate inducible DNA sequences from the CarG elements of the cancer cells.73–75 Egr1 promoter have been cloned upstream of a cDNA An important feature of the suppressor network in encoding TNFa and expressed as an adenoviral construct. prostate tumors is Egr1-induced TGFb1.61,63,64 TGFb1 Thus, expression of either an anti-Egr1 factor or a general

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