Egr1 Promotes Growth and Survival of Prostate Cancer Cells

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. Furthermore we also showed that Egr1 is responsible for cyclin D2 overexpression in tumorigenic DU145 human prostate cells. The regulation of these genes by Egr1 was demonstrated using Egr1 antisense oligonucleotides that further implicated Egr1 in resistance to apoptotic signals. One mechanism was illustrated by the ability of Egr1 to inhibit CD95 (Fas/Apo) expression, leading to insensitivity to FasL. The results provide a mechanistic basis for the oncogenic role of Egr1 in TRAMP C2 prostate cancer cells. 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. Furthermore we also showed that Egr1 is responsible for cyclin D2 overexpression in tumorigenic DU145 human prostate cells. The regulation of these genes by Egr1 was demonstrated using Egr1 antisense oligonucleotides that further implicated Egr1 in resistance to apoptotic signals. One mechanism was illustrated by the ability of Egr1 to inhibit CD95 (Fas/Apo) expression, leading to insensitivity to FasL. The results provide a mechanistic basis for the oncogenic role of Egr1 in TRAMP C2 prostate cancer cells. transforming growth factor antisense oligonucleotide control oligonucleotide reverse transcriptase inhibitor κBα Prostate cancer is the most common malignancy in men and a frequent cause of cancer death. The mortality of this disease is due to metastasis to the bone and lymph nodes. Prostate cancer progression is thought to proceed from multiple defined steps through prostatic intra-epithelial neoplasia, invasive cancer, and progression to androgen-independent and refractory terminal phase (44Nagle R.B. Petein M. Brawer M. Bowden G.T. Cress A.E. J. Cell. Biochem. 1992; 16 (suppl.): 26-29Google Scholar, 50Scher H.I. Heller G. Urology. 2000; 55: 323-327Google Scholar). A large fraction of early onset, and up to 5–107 of all prostate cancer patients, may have an inherited germline mutation that has facilitated the onset of carcinogenesis. However, in the majority of cases, no inherited gene defects are involved, and cancer arises as a result of a series of acquired somatic genetic changes affecting many genes on several chromosomes. Although the molecular mechanism of prostate cancer progression remains largely unknown, a few genes such as E-cadherin, α-catenin, TGF-ॆ,1 and insulin-like growth factors I and II have been shown to be aberrantly expressed and are markers of prostate cancer (34Lamharzi N. Schally A.V. Koppan M. Regul. Pept. 1998; 77: 185-192Google Scholar, 69Wolk A. Mantzoros C.S. Andersson S.O. Bergstrom R. Signorello L.B. Lagiou P. Adami H.O. Trichopoulos D. J. Natl. Cancer Inst. 1998; 90: 911-915Google Scholar). To clearly understand the multistep progression of this disease many other genes remain to be identified. One of the overexpressed genes found in prostate cancer tissue is the transcription factor early growth response gene 1 (Egr1) (18Eid M.A. Kumar M.V. Iczkowski K.A. Bostwick D.G. Tindall D.J. Cancer Res. 1998; 58: 2461-2468Google Scholar, 62Thigpen A.E. Cala K.M. Guileyardo J.M. Molberg K.H. McConnell J.D. Russell D.W. J. Urol. 1996; 155: 975-981Google Scholar). This gene could have an important function because its expression level increases with the degree of malignancy as measured by the Gleason grade of the tumor (18Eid M.A. Kumar M.V. Iczkowski K.A. Bostwick D.G. Tindall D.J. Cancer Res. 1998; 58: 2461-2468Google Scholar). This seems to be specific to prostate tumor cells, because in mammary and lung tumors, as well as most normal tissues, Egr1 expression is low. Egr1 overexpression is correlated with the loss of its co-repressor NAB2 in primary prostate carcinoma. This disruption of the balance between Egr1 and NAB2 expression results in a high Egr1 transcriptional activity in prostate carcinoma cells (1Abdulkadir S.A. Carbone J.M. Naughton C.K. Humphrey P.A. Catalona W.J. Milbrandt J. Hum. Pathol. 2001; 32: 935-939Google Scholar). A recent study based on the cross breeding of Egr1−/− mice with TRAMP mice showed significantly delayed prostate tumor formation in the Egr1-deficient TRAMP mouse compared with TRAMP-Egr1+/+ mice (2Abdulkadir S.A. Qu Z. Garabedian E. Song S.K. Peters T.J. Svaren J. Carbone J.M. Naughton C.K. Catalona W.J. Ackerman J.J. Gordon J.I. Humphrey P.A. Milbrandt J. Nat. Med. 2001; 7: 101-107Google Scholar). The TRAMP mouse is a well known model of prostate cancer (20Foster B.A. Gingrich J.R. Kwon E.D. Madias C. Greenberg N.M. Cancer Res. 1997; 57: 3325-3330Google Scholar) in which tumors progress to metastases in a window from 8 to 24 weeks of age. Although Egr1 loss did not appear to prevent tumor initiation, Egr1 deficiency delayed the progression of prostate tumors in these mice. Significantly, several gene products associated with aggressive prostate cancer such as TGF-ॆ and insulin-like growth factor II (37Liu C. Adamson E. Mercola D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11831-11836Google Scholar, 60Svaren J. Ehrig T. Abdulkadir S.A. Ehrengruber M.U. Watson M.A. Milbrandt J. J. Biol. Chem. 2000; 275: 38524-38531Google Scholar) have been identified as regulated by Egr1. These observations strongly suggest that Egr1 is involved in prostate cancer progression despite its known role as a tumor-suppressor in several other types of human cancers (29Huang R. Fan Y. de Belle I. Niemeyer C. Gottardis M. Mercola D. Adamson E. Int. J. Cancer. 1997; 72: 102-109Google Scholar). In this present study on the role of Egr1, we have used the tumorigenic C2 prostate cancer cell line which was established from a prostate tumor from a single TRAMP mouse tumor. These tumorigenic cells express a high constitutive level of Egr1 protein. Transcriptional regulation by Egr1 was assessed using Affymetrix array technology. The unique step used here was to perform a microarray analysis using cells rendered deficient in Egr1 as the comparison sample for the identification of Egr1 target genes, in prostate cancer cells. The results provide new insight into the involvement of endogenous Egr1 in proliferation and survival of prostate cancer cells by the identification of several new target genes specifically controlling growth, cell cycle progression, and the apoptosis pathway. C2 TRAMP cells were grown as described elsewhere (20Foster B.A. Gingrich J.R. Kwon E.D. Madias C. Greenberg N.M. Cancer Res. 1997; 57: 3325-3330Google Scholar). The cells were seeded into 35-mm dishes at a density of 100,000 cells per well 1 day before transfection. The transfection was performed as described by the manufacturer with the GenePorter reagent (16 ॖl) (Gene Therapy Systems, Inc, San Diego, CA) and 0.1 ॖm antisense oligonucleotide (AS or ctl). Sequences of the AS and mismatch control oligonucleotide (ctl) were used as described (65Virolle T. Adamson E.D. Baron V. Birle D. Mercola D. Mustelin T. de Belle I. Nat. Cell Biol. 2001; 3: 1124-1128Google Scholar). The sequence of ctl oligonucleotide corresponds to AS sequence with 4 bases mutated. One day before transfection the cells were seeded in duplicate into 35-mm dishes at a density of 70,000 cells per dish. At day 0 cells were transfected as described above. 4 h later the cells were harvested for counting and for protein and total mRNA extraction. This procedure was repeated each day after transfection according to a time course from day 0 to day 6. The day after transfection, the cells were ultraviolet-C (UVC) irradiated (40 J/m2) in a Stratalinker (Stratagene, La Jolla, CA) or treated with 100 ng/ml of Fas L recombinant protein (Oncogene Research Products, Darmstadt, Germany). One or two days after UVC irradiation or 9 and 18 h after Fas L treatment, detached and trypsinized cells were pooled and incubated with 0.27 trypan blue to determine the percentage of dead cells. C2 cells were transfected as described above. After 16 h the cells were counted and seeded into 6 well plates (200 cells/well) in RPMI medium with 0.1 ॖm of antisense oligonucleotide. After 8 days incubation at 37 °C, the colonies were stained with 27 crystal violet. The protocol recommended by Affymetrix (www.affymetrix.com) was used for mRNA control and gene expression analysis from C2 cells transfected with AS or ctl The were to Affymetrix mouse for analysis and of the and other of the microarray analysis using Affymetrix were used as D.J. M.V. M. C. M. Nat. 1996; Scholar). mRNA expression level was by real-time using the I according to the A from several of a sample of total was established to the of each were to the of from a gene was using the specific the were and with to Egr1 mouse and human cyclin D2 and or CD95 Systems, To protein on the cells were incubated in at 4 After as described elsewhere Belle I. Mercola D. Adamson E.D. 2000; the chromatin was by to an of The was using a specific Egr1 and a as a After as described elsewhere Belle I. Mercola D. Adamson E.D. 2000; the for identification of the sequence of Egr1 target genes was performed by using the in the of the and and from the cyclin D2, and and from the cyclin and and from the and and from the and and from the was used as a control for the of each and from C2 cells were used as The products were on To the of Egr1 overexpression in prostate cancer cells, we its expression using an AS in TRAMP C2 prostate cancer cells. To the and the of we performed of the protein expression of Egr1 and other and 24 h after transfection of the antisense and control oligonucleotides in the antisense oligonucleotide strongly Egr1 expression, was no on and seems to be Egr1 was In the ctl did not the protein expression of the cells. These results demonstrated that a with a 0.1 of the AS oligonucleotide and specifically Egr1 To the time course of Egr1 in C2 cells, were each day for 6 days AS transfection of antisense and control cells. Egr1 expression in the of AS was from day 1 to day on day and was on day to day 6 the of the ctl did not Egr1 expression level These results that AS is days to and specific of Egr1 expression for a a single To determine the involvement of Egr1 in the proliferation of C2 cells, the growth of the cells in which Egr1 expression was by AS oligonucleotide was compared with the control to C2 cells transfected with control oligonucleotide the cells were transfected at day 0 with AS or ctl and the proliferation was assessed day day 6 by cell counting in the proliferation of cells was strongly the days after transfection and to on day day 4 and the of the proliferation was to the of the control that the cells proliferation The proliferation time course was well correlated to the of Egr1 in as as Egr1 expression was the proliferation of C2 cells was and as as Egr1 expression In comparison between and cells in a showed colonies in of colonies for colonies for that the of the cells may Egr1 is cell cycle analysis by cell performed at day after transfection, showed cells in the phase of cells cells not The of results strongly in of a role for Egr1 in the control of growth and cell cycle progression in prostate cancer cells. To determine the genes that are involved in of mRNA from C2 cells 1 day after transfection with AS or with ctl oligonucleotide were performed using Affymetrix microarray Affymetrix analysis a large of genes involved in the control of and of these not been identified as of an Egr1 pathway. Although many genes are Egr1 target genes, could be regulated by Egr1 or after the of the of the cells due to the of Egr1 is important to that genes could be as important as the target genes to or Egr1 the Affymetrix expression changes with AS are in analysis of genes regulated in C2 cells that express Egr1 compared with antisense treated by target A of proliferation R. S. M. C. M. A. M. Pept. 1996; protein of proliferation E. E. C. A. C. E. M. C. G. E. P. M. J. 2001; and cycle at Cell. Biol. Scholar, C. A. S. U. 1997; of cell growth C. N. S. G. M. is high in prostate cancer cell J. Bostwick D.G. J.M. T.J. R.B. Pathol. of proliferation 1998; of to apoptosis S. 2001; Scholar, S. M. J. 2001; S. 2001; growth factor of growth J. V. S. G. P. S. C. M. Cell Biol. of cell cycle progression D. M. J. Biol. Chem. 1997; with of proliferation J. J. Biochem. Biol. J. J. Biochem. Biol. of apoptosis D. I. A. S. P. Cancer Res. 2001; D. I. A. S. P. Cancer Res. 2001; A. C. J. 1997; cycle at J.D. Cell Biol. Int. 2001; Scholar, J. M. M. A. 2001; protein for apoptosis after Cell. 1997; A. Cancer Res. 2001; inhibitor of of cell proliferation T. J. Biol. Chem. Scholar) and of apoptosis J. Biol. Chem. 2001; M. J. M. 2001; A. T. T. A. Biochem. Res. 2000; of apoptosis M. M. Z. C. R. I. Cancer Res. M. M. J. Urol. 2000; protein of apoptosis G. J. C. G. J. D. Greenberg A. J. Biol. Chem. of apoptosis Z. Y. M. S. Proc. Natl. Acad. Sci. U. S. A. 1997; by response by and growth factor Cell. Biol. lung cell proliferation and involved in 1996; Scholar, R. J. 2001; with Egr1 expression R. J. 2001; in tumors V. J. Cancer. involved in cell cycle progression G. G. D. D. Biol. protein proliferation A. A. R. M.A. J. Cell. of apoptosis P. E. J. Biol. Chem. 1996; of cell proliferation and of cell cycle progression J. 2001; Scholar, B.A. A. C. T. M. R. Cancer Res. 2001; with Egr1 expression R. G. J. 2000; of survival through activity A. Y. Y. Y. Y. J. Pathol. 2001; associated in Y. J. N. 2000; growth proliferation M.A. M. C. 1996; growth factor protein in prostate tumor cell involved in of proliferation C. 2001; C. 2001; level expression in prostate tumor cells G. J. C. G. J. D. Greenberg A. J. Biol. Chem. Scholar, A. A. R. M.A. J. Cell. P. J. A. Res. 2001; Scholar, C. T. C. D. A. C. Res. 2000; C. J. de Belle I. Adamson E.D. Mercola D. J. Biol. Chem. each the its function involvement in human prostate cancer with prostate and on its regulation by Egr1 as Egr1 target are in a new each the its function involvement in human prostate cancer with prostate and on its regulation by Egr1 as Egr1 target are To the Affymetrix microarray analysis the expression of genes in I was by real-time In these total from Egr1 and C2 cells were used as The from real-time compared with the in the Affymetrix analysis The or of specific target genes by Egr1 was in the in all and a degree of the of the Affymetrix and real-time results was which is These results the of the Affymetrix of Affymetrix array with real-time for mRNA in the expression level of several Egr1 target genes in I were using analysis of from and treated cells. The results were to and expressed as the of were performed in from two and the are also and and in response to Egr1 a by in a new in the expression level of several Egr1 target genes in I were using analysis of from and treated cells. The results were to and expressed as the of were performed in from two and the are also and and in response to Egr1 a by of I several genes identified as Egr1 such as transforming growth factor 1 (37Liu C. Adamson E. Mercola D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11831-11836Google Scholar, 60Svaren J. Ehrig T. Abdulkadir S.A. Ehrengruber M.U. Watson M.A. Milbrandt J. J. Biol. Chem. 2000; 275: 38524-38531Google Scholar) and CD95 A. C. J. 1997; Scholar). of other genes such as transcription factor and cyclin D2 is known to be correlated with an of Egr1 expression R. G. J. 2000; Scholar, R. J. 2001; Scholar). several genes identified have been to human prostate inhibitor κBα was shown in several prostate cancer cell to inhibit growth, and metastasis by of activity S. 2001; Scholar). by with is known to prevent the transforming of by of the C. N. S. G. M. Scholar). In is found to be overexpressed in prostate cancer cells J. Bostwick D.G. J.M. T.J. R.B. Pathol. Scholar). Egr1 could by of is associated with of cell proliferation in the human prostate cancer cell line J. J. Biochem. Biol. Scholar). of this protein is in prostate cancer cells and be by and other factors involved in the control of cell proliferation J. J. Biochem. Biol. Scholar). and CD95 are known to be involved in the apoptotic response in prostate cancer cell D. I. A. S. P. Cancer Res. 2001; Scholar, M. M. Z. C. R. I. Cancer Res. Scholar). all these genes, which as tumor are by Egr1 in C2 prostate cancer cells. the other genes which cell proliferation in and human tumor prostate cells C. 2001; and which is strongly expressed in prostate cancer cells P. J. A. Res. 2001; Scholar, C. T. C. D. A. C. Res. 2000; are by Egr1. In the genes that are involved in cell cycle progression, or of apoptosis are all by Egr1, involved in growth and apoptosis are constitutive expression of Egr1 in prostate cancer the balance between survival and tumor in of Egr1 expression for days after AS transfection. a time course of expression be for Egr1 target mRNA expression of cyclin D2 and known to growth and cell cycle progression J. 2001; Scholar, cyclin which inhibit cell cycle progression Cell. Biol. Scholar, D. M. J. Biol. Chem. 1997; were measured from day 0 to day 6 by real-time D2 and mRNA expression was from day 1 to day Egr1 was and as as Egr1 expression was normal of cyclin and was day Egr1 expression was and normal expression was on day These results were not with the ctl that Egr1 expression is for mRNA expression of cyclin D2 and and to and cyclin mRNA The of this regulation that no other transcription factor for the of Egr1 function in these cells. To mRNA regulation by Egr1 is at the protein analysis was performed on from and cells from day 0 to day 6 after transfection. In these cyclin D2 and expression level was The results showed a of cyclin D2 and an of protein expression in antisense treated cells, which the time course of mRNA expression These results that Egr1 by antisense is to Egr1 target gene expression at the protein In the cyclin D2 and protein expression day 0 to day are also correlated to the found in the cell cycle analysis and in the proliferation between and cells. This corresponds to in the regulation of cell cycle cyclin are for cell cycle progression and overexpression of is responsible for the phase 1996; Cell. Scholar). cyclin D2 is found to be by Egr1, expression, a cyclin inhibitor J. M. E. J. 2000; is Egr1, by the of and cyclin D2, cell cycle progression and a role in prostate cancer cells. To the of these we cyclin D2, and protein expression by real-time in the human prostate cancer cell transfected with AS or ctl oligonucleotide. in C2 cells, Egr1 expression is high in DU145 and strongly by the antisense oligonucleotide In Egr1 regulation of these genes to be the as the regulation in the C2 mouse model Furthermore cyclin D2 protein expression is also strongly the of Egr1 expression, that Egr1 is to cyclin D2 protein expression level in DU145 as well as in mouse TRAMP C2 cells To Egr1 and cyclin D2 expression human prostate cancer progression, we cell normal prostate cells, tumorigenic cells, and tumorigenic DU145 human prostate cells. Egr1 expression is in normal human prostate and cell is overexpressed in cyclin D2 expression with Egr1 expression in these cell and is strongly expressed in the aggressive tumorigenic DU145 cells These results the of C2 cells as a model to new Egr1 target genes in prostate Egr1 may also a role in prostate cancer by affecting prostate cell survival C. Fan Y. Mercola D. Adamson E.D. Cancer Res. 55: Scholar) or apoptosis (65Virolle T. Adamson E.D. Baron V. Birle D. Mercola D. Mustelin T. de Belle I. Nat. Cell Biol. 2001; 3: 1124-1128Google and this was and cells were and dead cells were counted by trypan blue 24 and h of the cells were dead 24 h of cells were dead at h of control for cells These a role for Egr1 in response to endogenous expression of Egr1 is not for proliferation of C2 cells also to to a of human prostate cancer cells S. Urol. 2000; Scholar). Affymetrix analysis several genes that are by Egr1, such as C. Cell Scholar, M. M. Z. C. R. I. Cancer Res. protein G. J. C. G. J. D. Greenberg A. J. Biol. Chem. Scholar) and CD95 D. I. A. S. P. Cancer Res. 2001; a gene involved in apoptosis a of tumor factor is as because of its ability to signals. the other the gene by Egr1, is involved in of apoptosis P. E. 1996; Scholar, P. E. J. Biol. Chem. 1996; a role for Egr1 as in prostate cancer cells. Egr1 regulation of confirmed at the mRNA level by real-time was also for protein expression in and cells treated or not by UVC In cells, UVC to a of Egr1 expression, which was strongly by AS CD95 expression to be in cells was clearly expressed in cells After UVC CD95 expression was strongly in was expressed in These results at the protein level the of CD95 expression by Egr1. This mechanism of is all the is after a To this in CD95 expression could be as to Fas L we treated and cells for 9 and 18 h with Fas L and counted the percentage of dead cells by trypan blue from CD95 protein expression were to Fas at 9 h after treatment, of cells were dead in in cell This in the resistance to cell between and cells, was present after 18 h treatment, with of dead cells compared with high constitutive Egr1 expression apoptosis of prostate cancer cells by Fas in by CD95 The of the CD95 in prostate apoptosis has also been demonstrated in the normal prostate A. A. E. A. R. Scholar). In further have demonstrated the involvement of CD95 in prostate cancer cells to apoptosis after or irradiation J. Cancer. Scholar, J. Biol. Chem. 2000; 275: Scholar). These results well a of Egr1 in the cell response and suggest that to apoptosis by the of Egr1 expression could an Furthermore this that the of gene expression by Egr1 is a in the of prostate cancer cells. and real-time are and to the expression between two mRNA not determine the regulation by Egr1 or we performed chromatin and immunoprecipitation to of of Egr1 target genes by the Affymetrix this AS and ctl C2 cells were After chromatin in cells, Egr1 to its These target were by specific Egr1 immunoprecipitation and immunoprecipitation was used as the control and C2 was used to of each were to specifically of cyclin and cyclin D2, to in the by sequence analysis of each of these genes showed several Egr1 and and cyclin D2 an from and ctl that showed the as the control cyclin was not A and no was found for the control and the AS these results that the were by Egr1 in the regulation of and cyclin D2 by Egr1. to the that these genes could be regulated in of the of the we performed a study of the regulation of a well known Egr1 target gene C. J. de Belle I. Adamson E.D. Mercola D. J. Biol. Chem. Scholar). AS oligonucleotide is at h after transfection not we performed the analysis at and for the of cyclin D2 and at h after AS to the onset of Egr1 these results that many of the Egr1 target genes identified in study may be regulated by Egr1. study new insight on the and of Egr1 in prostate cancer cells. We that Egr1 cell growth and to by a of genes known to be important in cell cycle progression, growth, and constitutive Egr1 expression here in prostate cancer cells is to tumor cell growth and We suggest that results the of Milbrandt and (2Abdulkadir S.A. Qu Z. Garabedian E. Song S.K. Peters T.J. Svaren J. Carbone J.M. Naughton C.K. Catalona W.J. Ackerman J.J. Gordon J.I. Humphrey P.A. Milbrandt J. Nat. Med. 2001; 7: 101-107Google Scholar) in that the mechanistic basis of the role of Egr1 in cancer growth as well as study for the time in the growth role of Egr1 in other such and tumor cells J. Cell. Biochem. Scholar, V. J. N. 2000; Scholar). However, these are because in cancer, and Egr1 as a tumor gene A. A. G. A. Mercola D. C. V. M. G. R. A. G. Cancer Res. 2001; 7: Scholar, R. Fan Y. de Belle I. Niemeyer C. Gottardis M. Mercola D. Adamson E. Int. J. Cancer. 1997; 72: 102-109Google Scholar) that be for to irradiation S. M. J. Biol. Chem. 1996; Scholar, T. Adamson E.D. Baron V. Birle D. Mercola D. Mustelin T. de Belle I. Nat. Cell Biol. 2001; 3: 1124-1128Google Scholar). of the and the regulation of Egr1 target genes from these this