KLF6 Induces Apoptosis in Prostate Cancer Cells through Up-regulation of ATF3

KLF6 (Kruppel-like factor 6) is a zinc finger transcription factor and a tumor suppressor that is frequently mutated in prostate cancer. KLF6 suppresses tumor growth and induces apoptosis in cancer cells through mechanisms still not defined. Here we show that KLF6 induces apoptosis in prostate cancer cells by ATF3 (activating transcription factor 3) expression. KLF6 binds directly to and activates the ATF3 promoter. ATF3 induced apoptosis when ectopically expressed in cells, whereas knockdown of ATF3 by small interference RNA blocked KLF6-induced apoptosis. KLF6 mutants derived from clinical prostate cancers failed to activate the ATF3 promoter and were unable to induce apoptosis. Furthermore, stress conditions (exposure to staurosporine and hypoxia induced by sodium azide) caused significant increase in ATF3 expression and induced apoptosis, whereas knockdown of KLF6 by small interference RNA blocked the increase of ATF3 as well as the induction of apoptosis in these conditions. Thus, ATF3 is a key mediator of KLF6-induced apoptosis in prostate cancer cells. KLF6 (Kruppel-like factor 6) is a zinc finger transcription factor and a tumor suppressor that is frequently mutated in prostate cancer. KLF6 suppresses tumor growth and induces apoptosis in cancer cells through mechanisms still not defined. Here we show that KLF6 induces apoptosis in prostate cancer cells by ATF3 (activating transcription factor 3) expression. KLF6 binds directly to and activates the ATF3 promoter. ATF3 induced apoptosis when ectopically expressed in cells, whereas knockdown of ATF3 by small interference RNA blocked KLF6-induced apoptosis. KLF6 mutants derived from clinical prostate cancers failed to activate the ATF3 promoter and were unable to induce apoptosis. Furthermore, stress conditions (exposure to staurosporine and hypoxia induced by sodium azide) caused significant increase in ATF3 expression and induced apoptosis, whereas knockdown of KLF6 by small interference RNA blocked the increase of ATF3 as well as the induction of apoptosis in these conditions. Thus, ATF3 is a key mediator of KLF6-induced apoptosis in prostate cancer cells. KLF6 (Kruppel-like factor 6) is a tumor suppressor protein that is down-regulated or mutated in several types of cancers, including prostate cancer (1Ito G. Uchiyama M. Kondo M. Mori S. Usami N. Maeda O. Kawabe T. Hasegawa Y. Shimokata K. Sekido Y. Cancer Res. 2004; 64: 3838-3843Crossref PubMed Scopus (135) Google Scholar, 3Kremer-Tal S. Reeves H.L. Narla G. Thung S.N. Schwartz M. Difeo A. Katz A. Bruix J. Bioulac-Sage P. Martignetti J.A. Friedman S.L. Hepatology. 2004; 40: 1047-1052Crossref PubMed Scopus (122) Google Scholar). KLF6 is a zinc finger transcription factor that binds to a GC box and regulates the expression of target genes. It has been shown that KLF6 suppresses tumor growth through activating p21WAF1/Cip1, an inhibitor of the cyclin-dependent kinases, in both cultured cells and a transgenic mouse model (2Narla G. Heath K.E. Reeves H.L. Li D. Giono L.E. Kimmelman A.C. Glucksman M.J. Narla J. Eng F.J. Chan A.M. Ferrari A.C. Martignetti J.A. Friedman S.L. Science. 2001; 294: 2563-2566Crossref PubMed Scopus (381) Google Scholar, 4Narla G. Kremer-Tal S. Matsumoto N. Zhao X. Yao S. Kelley K. Tarocchi M. Friedman S.L. Oncogene. 2007; 26: 4428-4434Crossref PubMed Scopus (69) Google Scholar). KLF6 also directly interacts with cyclin D1 to suppress cyclin-dependent kinase 4 and causes cell cycle arrest (5Benzeno S. Narla G. Allina J. Cheng G.Z. Reeves H.L. Banck M.S. Odin J.A. Diehl J.A. Germain D. Friedman S.L. Cancer Res. 2004; 64: 3885-3891Crossref PubMed Scopus (131) Google Scholar). Besides growth inhibition, KLF6 has also been shown to induce apoptosis in non-small cell lung cancer cells (1Ito G. Uchiyama M. Kondo M. Mori S. Usami N. Maeda O. Kawabe T. Hasegawa Y. Shimokata K. Sekido Y. Cancer Res. 2004; 64: 3838-3843Crossref PubMed Scopus (135) Google Scholar). However, the mechanism of KLF6-induced apoptosis is still not known. ATF3 (activating transcription factor 3) is a member of the ATF/cAMP-response element-binding protein family of transcription factors (6Liang G. Wolfgang C.D. Chen B.P. Chen T.H. Hai T. J. Biol. Chem. 1996; 271: 1695-1701Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). ATF3 is rapidly up-regulated under various stress conditions, including hepatotoxicity, UV/ionizing radiation, and exposure to DNA-damaging agents (7Hai T. Hartman M.G. Gene (Amst.). 2001; 273: 1-11Crossref PubMed Scopus (659) Google Scholar). ATF3 can also be induced by ischemia and hypoxia (8Allen-Jennings A.E. Hartman M.G. Kociba G.J. Hai T. J. Biol. Chem. 2001; 276: 29507-29514Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 9Chen B.P. Wolfgang C.D. Hai T. Mol. Cell. Biol. 1996; 16: 1157-1168Crossref PubMed Scopus (259) Google Scholar, 10Lu D. Wolfgang C.D. Hai T. J. Biol. Chem. 2006; 281: 10473-10481Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). ATF3 is a pro-apoptotic protein. It induces apoptosis in ovarian cancer cells (11Syed V. Mukherjee K. Lyons-Weiler J. Lau K.M. Mashima T. Tsuruo T. Ho S.M. Oncogene. 2005; 24: 1774-1787Crossref PubMed Scopus (96) Google Scholar) and enhances etoposide- or camptothecin-induced apoptosis in HeLa cells (12Mashima T. Udagawa S. Tsuruo T. J. Cell. Physiol. 2001; 188: 352-358Crossref PubMed Scopus (92) Google Scholar). Although transgenic mice expressing ATF3 in beta cells develop abnormal islets and defects secondary to beta cell apoptosis, primary islets derived from ATF3 knock-out mice were partially protected from cytokine- and nitric oxide-induced apoptosis (13Hartman M.G. Lu D. Kim M.L. Kociba G.J. Shukri T. Buteau J. Wang X. Frankel W.L. Guttridge D. Prentki M. Grey S.T. Ron D. Hai T. Mol. Cell. Biol. 2004; 24: 5721-5732Crossref PubMed Scopus (259) Google Scholar). Additionally, fibroblasts from ATF3 knock-out mice were partially protected from UV-induced apoptosis (10Lu D. Wolfgang C.D. Hai T. J. Biol. Chem. 2006; 281: 10473-10481Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). On the other hand, it was shown that ATF3 overexpression promoted invasiveness of prostate tumor cells in vitro and significantly enhanced spontaneous lung metastasis without affecting primary tumorigenicity in a severe combined immunodeficient mouse model (14Bandyopadhyay S. Wang Y. Zhan R. Pai S.K. Watabe M. Iiizumi M. Furuta E. Mohinta S. Liu W. Hirota S. Hosobe S. Tsukada T. Miura K. Takano Y. Saito K. Commes T. Piquemal D. Hai T. Watabe K. Cancer Res. 2006; 66: 11983-11990Crossref PubMed Scopus (93) Google Scholar). Interestingly, it was also shown that ATF3 has a dichotomous role in cancer development, while enhancing apoptosis in the untransformed MCF10A cells, ATF3 inhibited apoptosis in the more aggressive MCF10CA1a cells and enhanced cell mobility (15Yin X. Dewille J.W. Hai T. Oncogene. 2008; 27: 2118-2127Crossref PubMed Scopus (160) Google Scholar). We found that KLF6 induces apoptosis when ectopically expressed in prostate cancer cells. To understand the mechanism of KLF6-induced apoptosis, we employed microarray gene expression analysis and identified ATF3 as one of the target genes regulated by KLF6. We further demonstrated that ATF3 is a key mediator of KLF6-induced apoptosis in prostate cancer cells. KLF6 and ATF3 are required for cancer cell apoptosis in stress conditions. Cells and Transfection—Human prostate cancer cell lines PC-3 and LNCaP were purchased from American Type Culture Collection. The cells were cultured in RPMI 1640 media containing 10% fetal bovine serum. For transient transfection, plasmids were transfected into cells using Lipofectamine™Plus reagent (Invitrogen) following the manufacturer's protocol. Drugs and Chemicals—Staurosporine and sodium azide were purchased from Sigma. Staurosporine was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 mm. Sodium azide was dissolved in PBS 3The abbreviations used are: PBS, phosphate-buffered saline; siRNA, small interference RNA; ChIP assay, chromatin immunoprecipitation assay; EMSA, electrophoretic mobility shift assay; KB, KLF6 binding region; PARP, poly(ADP-ribose) polymerase. at concentration of 4 m. In all studies, an equivalent amount of diluent (DMSO or PBS) was added to the culture media as a negative control. Plasmid Construction—Human cDNAs encoding full-length KLF6 gene and SV2 variant were obtained by PCR amplification using an EST clone (I.M.A.G.E. clone ID 3623401) as template. KLF6 and KLF6-SV2 cDNAs were subcloned into pCMV-Tag2 (Stratagene) vector to express a FLAG-tagged protein. For doxycycline-inducible expression using the Tet-On advanced system, KLF6 was subcloned into pTRE-Tight vector (Clontech). The pCG and pCG-ATF3 expression vectors were kindly provided by Dr. Tsonwin Hai at Ohio State University. The ATF3 promoter reporter plasmids Luc-1850, Luc-632, Luc-111, and Luc-84 were kindly provided by Dr. Shigetaka Kitajima of Tokyo Medical and Dental University. Luc-258 was generated by PCR. Luc-632 and Luc-258 carrying KLF6-binding site mutants (CC to AA) and KLF6 mutants were created by PCR using the QuickChange II site-directed mutagenesis kit (Stratagene), following the supplied protocol. Cell Viability and Apoptosis Assays—Trypan blue dye exclusion assay was used to measure cell viability. Briefly, cells were suspended 1:1 with 0.4% trypan blue solution. Dead cells (stained blue) were counted under a microscope using a hemocytometer. Apoptosis was measured using the Cell Death Detection ELISAPLUS kit (Roche Applied Science) following the manufacturer's protocol. This assay determines apoptosis by measuring mono- and oligonucleosomes in the lysates of apoptotic cells. The cell lysates were placed into a streptavidin-coated microplate and incubated with a mixture of anti-histone/biotin and anti-DNA/peroxidase. The amount of peroxidase retained in the immunocomplex was photometrically determined with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid as the substrate. Absorbance was measured at 405 nm. Western Blot Analysis—Cells were lysed in RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS). Complete protease inhibitor mixture (Roche Applied Science) was added to lysis buffer before use. Protein concentration was determined by the DC protein assay (Bio-Rad). Protein samples were subjected to SDS-PAGE and transferred to nitrocellulose membrane. The membrane was blocked in 5% nonfat milk in PBS overnight and incubated with primary antibody and subsequently with appropriate horseradish peroxidase-conjugated secondary antibody. Signals were developed with ECL reagents (Amersham Biosciences) and exposure to x-ray films. Anti-ATF3 polyclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-β-tubulin monoclonal antibody was purchased from Sigma. Anti-KLF6 monoclonal antibody was purchased from Invitrogen. Anti-KLF6 polyclonal antibody was purchased from Santa Cruz Biotechnology. Anti-cleaved caspase-9 and anti-cleaved PARP polyclonal antibodies were purchased from Cell Signaling Technologies (Danvers, MA). siRNAs and Transfection—Silencer™ pre-designed siRNAs and negative control siRNA (catalog number 4611) were purchased from The for KLF6 siRNA and The for ATF3 siRNA is and The for siRNA is and The siRNAs were transfected into PC-3 cells using siRNA reagent (Roche Applied Science) following the manufacturer's protocol. Cells were cultured and transfected in cells and the siRNA were nm. Protein samples were for Western of ATF3 was measured by PCR using gene expression assay (catalog number from Applied CA). RNA was from PC-3 cells using kit of RNA was used in transcription The cDNAs were used as to PCR an Applied PCR following the manufacturer's protocol. or LNCaP cells in were with reporter plasmids and or expression To of the ATF3 promoter K. S. J. M. M. Y. Y. M. Kitajima S. J. 2005; 24: PubMed Scopus Google cells were incubated in media containing 0.1% inhibitor was added to the culture media to cell caused by KLF6 expression. of cell were for using a assay assay was using the ChIP assay kit from following the supplied protocol. were using or control PCR was with the following for the ATF3 an from to as the negative and and KLF6 binding from to and KLF6 protein was incubated with the following for was using following the manufacturer's protocol. the were for using the for was to be KLF6 Apoptosis in PC-3 and LNCaP ectopically KLF6 induced apoptosis in non-small cell lung cancer cells (1Ito G. Uchiyama M. Kondo M. Mori S. Usami N. Maeda O. Kawabe T. Hasegawa Y. Shimokata K. Sekido Y. Cancer Res. 2004; 64: 3838-3843Crossref PubMed Scopus (135) Google Scholar). KLF6 is frequently mutated in prostate cancer (2Narla G. Heath K.E. Reeves H.L. Li D. Giono L.E. Kimmelman A.C. Glucksman M.J. Narla J. Eng F.J. Chan A.M. Ferrari A.C. Martignetti J.A. Friedman S.L. Science. 2001; 294: 2563-2566Crossref PubMed Scopus (381) Google Scholar, X. J. Full Text Full Text PDF PubMed Scopus Google we determined KLF6 also induce apoptosis in prostate cancer cells. KLF6 was in PC-3 prostate cancer cells by transient using expression vector KLF6 expression significantly the of PC-3 cells PC-3 cells through apoptosis as by the assay measuring mono- and oligonucleosomes in the lysates of apoptotic cells and Western of PARP and caspase-9 of KLF6 also induced apoptosis in prostate cancer cell LNCaP cells and a pro-apoptotic of KLF6 in prostate cancer cells. KLF6 the of the mechanism of KLF6-induced apoptosis in prostate cancer cells, we the gene expression pCMV-Tag2 vector and PC-3 cells using microarray the genes that were up-regulated by KLF6 we ATF3 for further of in apoptosis (10Lu D. Wolfgang C.D. Hai T. J. Biol. Chem. 2006; 281: 10473-10481Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, M.G. Lu D. Kim M.L. Kociba G.J. Shukri T. Buteau J. Wang X. Frankel W.L. Guttridge D. Prentki M. Grey S.T. Ron D. Hai T. Mol. Cell. Biol. 2004; 24: 5721-5732Crossref PubMed Scopus (259) Google Scholar). We the of ATF3 expression by KLF6 using PCR. shown in overexpression of KLF6 by transient caused increase of ATF3 with cells transfected with Furthermore, KLF6 also caused significant in ATF3 protein expression in a as determined by Western of KLF6 also induced ATF3 expression in LNCaP cells the Tet-On gene expression we were to control the expression of KLF6 in PC-3 cells with shown in KLF6 expression at the induction of ATF3 protein expression at apoptosis was at the when the ATF3 protein was at not when KLF6 was induced at or KLF6 to and the ATF3 KLF6 directly regulates the expression of the ATF3 we an ATF3 gene reporter the promoter of the ATF3 gene promoter and the of the ATF3 promoter containing various shown in when KLF6 was expressed in LNCaP cells by with the reporter the ATF3 promoter was The to was still by KLF6. In further to and the of KLF6. The and the and in PC-3 cells, KLF6 was to activate the ATF3 and to and the of KLF6 the in the of to we identified a KLF6-binding site to that the of G. Y. D. J. V. 2006; PubMed Scopus Google Scholar). mobility shift assay that KLF6 can directly to whereas a to failed to with KLF6 To further KLF6 binds directly to promoter in we a chromatin immunoprecipitation assay, using PCR to target the ATF3 promoter In LNCaP cells KLF6 transient antibody was to the to of the ATF3 promoter not the and was obtained in PC-3 cells The control not with of the We mutated the KLF6-binding to and the of KLF6-induced ATF3 promoter To the role of we also created a reporter in the site is shown in in the of KLF6 was to activate the Luc-258 Luc-258 KLF6-binding reporter was not by KLF6. In the of of the KLF6-binding failed to KLF6 of the other transcription factors also activate promoter through binding to the ATF3 Apoptosis in PC-3 ATF3 a role in apoptosis in PC-3 cells, we ATF3 in PC-3 cells. of pCG-ATF3 plasmids into PC-3 cells in a significant increase in the ATF3 protein was by a significant induction of apoptosis in these cells Thus, ATF3 has pro-apoptotic in PC-3 cells. ATF3 KLF6-induced ATF3 is a factor that KLF6-induced apoptosis in PC-3 cells, we employed an siRNA to the expression of ATF3 in PC-3 cells. shown in ATF3 siRNA was to significantly the of ATF3 induction by KLF6 cells were transfected with vectors and control siRNA or ATF3 siRNA, and Although KLF6 induced apoptosis in cells transfected with the negative control siRNA, it failed to induce apoptosis in cells ATF3 was by siRNA KLF6 to ATF3 or been identified in as as of prostate cancer samples (2Narla G. Heath K.E. Reeves H.L. Li D. Giono L.E. Kimmelman A.C. Glucksman M.J. Narla J. Eng F.J. Chan A.M. Ferrari A.C. Martignetti J.A. Friedman S.L. Science. 2001; 294: 2563-2566Crossref PubMed Scopus (381) Google Scholar). shown that these KLF6 mutants the to cell a key of the KLF6 as a tumor suppressor (2Narla G. Heath K.E. Reeves H.L. Li D. Giono L.E. Kimmelman A.C. Glucksman M.J. Narla J. Eng F.J. Chan A.M. Ferrari A.C. Martignetti J.A. Friedman S.L. Science. 2001; 294: 2563-2566Crossref PubMed Scopus (381) Google Scholar). To the of KLF6 in ATF3 and apoptosis, we generated cDNAs encoding KLF6 mutants that been in clinical prostate cancer and (2Narla G. Heath K.E. Reeves H.L. Li D. Giono L.E. Kimmelman A.C. Glucksman M.J. Narla J. Eng F.J. Chan A.M. Ferrari A.C. Martignetti J.A. Friedman S.L. Science. 2001; 294: 2563-2566Crossref PubMed Scopus (381) Google Scholar). transfected into PC-3 cells, all KLF6 expressed at that of the KLF6 protein Although the KLF6 was to activate the ATF3 of the mutants was in the promoter reporter assay In to the of these was to ATF3 in PC-3 cells all KLF6 mutants failed to induce apoptosis when transfected into PC-3 cells KLF6 Apoptosis through of an role in stress conditions (6Liang G. Wolfgang C.D. Chen B.P. Chen T.H. Hai T. J. Biol. Chem. 1996; 271: 1695-1701Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, T. Hartman M.G. Gene (Amst.). 2001; 273: 1-11Crossref PubMed Scopus (659) Google Scholar). We that by ATF3 KLF6 a in apoptosis in prostate cancer cells. To we the of KLF6 knockdown by siRNA PC-3 cell apoptosis induced by agents as a protein kinase and sodium an of hypoxia to staurosporine or sodium azide induced significant in the of ATF3 protein in PC-3 cells were by the induction of apoptosis and To the role of KLF6 in PC-3 cell apoptosis in these conditions, we a KLF6 siRNA that was to KLF6 protein The KLF6 siRNA was to ATF3 induction caused by exposure to staurosporine or sodium as well as to apoptosis caused by these agents and Thus, KLF6 and target ATF3 in PC-3 cell apoptosis in stress conditions. KLF6 variant and are expressed in prostate cancer cells through G. Difeo A. Reeves H.L. J. E. Katz A. S. A. S.K. K.E. S.L. A.M. J. S.N. Friedman S.L. Martignetti J.A. Cancer Res. 2005; PubMed Scopus Google Scholar). The siRNA that we employed in the the expression of both KLF6 and the variant To the KLF6 also ATF3 we transfected PC-3 cells with shown in overexpression of KLF6-SV2 also induced ATF3 protein expression. KLF6-SV2 was also to induce apoptosis in PC-3 cells, to a with KLF6 Furthermore, using a siRNA G. A. Yao S. A. E. Reeves H.L. O. A. A. Chan A.M. Friedman S.L. Martignetti J.A. Cancer Res. 2005; PubMed Scopus Google we were to in PC-3 cells of by siRNA also blocked ATF3 as well as apoptosis in PC-3 cells The KLF6 gene is a in of prostate M. Cancer Res. 1996; Google Scholar, A.C. J.A. Cancer Res. 1996; Google Scholar). of KLF6 gene been identified in clinical prostate cancer samples (2Narla G. Heath K.E. Reeves H.L. Li D. Giono L.E. Kimmelman A.C. Glucksman M.J. Narla J. Eng F.J. Chan A.M. Ferrari A.C. Martignetti J.A. Friedman S.L. Science. 2001; 294: 2563-2566Crossref PubMed Scopus (381) Google Scholar, X. J. Full Text Full Text PDF PubMed Scopus Google a tumor suppressor role for KLF6 in prostate cancer. KLF6 expression has been shown to with clinical in prostate cancer W.L. J. 2004; PubMed Scopus Google Scholar). mechanism by KLF6 tumor is through of G. Kremer-Tal S. Matsumoto N. Zhao X. Yao S. Kelley K. Tarocchi M. Friedman S.L. Oncogene. 2007; 26: 4428-4434Crossref PubMed Scopus (69) Google a that is of (2Narla G. Heath K.E. Reeves H.L. Li D. Giono L.E. Kimmelman A.C. Glucksman M.J. Narla J. Eng F.J. Chan A.M. Ferrari A.C. Martignetti J.A. Friedman S.L. Science. 2001; 294: 2563-2566Crossref PubMed Scopus (381) Google Scholar). KLF6 also has pro-apoptotic in non-small cell lung cancer cells (1Ito G. Uchiyama M. Kondo M. Mori S. Usami N. Maeda O. Kawabe T. Hasegawa Y. Shimokata K. Sekido Y. Cancer Res. 2004; 64: 3838-3843Crossref PubMed Scopus (135) Google that apoptosis induction can be mechanism through KLF6 suppresses tumor However, the mechanism of apoptosis has not been defined. In we found that KLF6 also induced apoptosis when ectopically expressed in prostate cancer cells. Thus, apoptosis induction a key role in tumor in prostate that the of ATF3 expression and increase of ATF3 protein are the key mechanisms through KLF6 induces apoptosis in prostate cancer cells. We that not of ATF3 induce apoptosis in PC-3 cells, also of ATF3 by siRNA blocked KLF6-induced apoptosis. Furthermore, KLF6 mutants derived from clinical prostate cancers failed to activate ATF3 expression or to induce apoptosis, a role for the of the pro-apoptotic of KLF6 in clinical prostate cancer Although KLF6 the expression of other pro-apoptotic the siRNA knockdown a role of ATF3 in KLF6-induced apoptosis in prostate cancer cells. However, the mechanism of ATF3 regulates apoptosis in PC-3 cells to be Although LNCaP cells express of KLF6 and are PC-3 cells express of KLF6 and are However, were obtained in the promoter reporter assay and ChIP assay in the cell the of KLF6 ATF3 promoter is of the of expression. induces apoptosis by activating the expression of other pro-apoptotic as and T. Cell. Full Text PDF PubMed Scopus Google Scholar, J. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google in KLF6 and other in the of tumor by these transcription ATF3 is a stress is at in cells rapidly up-regulated by a of T. Wolfgang C.D. A.E. Gene Google Scholar). ATF3 induces apoptosis in stress conditions, and has been demonstrated in cultured cells as well as transgenic (11Syed V. Mukherjee K. Lyons-Weiler J. Lau K.M. Mashima T. Tsuruo T. Ho S.M. Oncogene. 2005; 24: 1774-1787Crossref PubMed Scopus (96) Google Scholar, M.G. Lu D. Kim M.L. Kociba G.J. Shukri T. Buteau J. Wang X. Frankel W.L. Guttridge D. Prentki M. Grey S.T. Ron D. Hai T. Mol. Cell. Biol. 2004; 24: 5721-5732Crossref PubMed Scopus (259) Google Scholar). We found that KLF6 and ATF3 a key role in PC-3 cell apoptosis caused by stress conditions, including staurosporine exposure and sodium of KLF6 by siRNA ATF3 induction and apoptosis in these stress conditions. In tumor cells are to conditions S. Chem. 2006; PubMed Scopus Google Scholar, P. 2004; PubMed Scopus Google Scholar). of of KLF6 gene the that were used in tumor cells a to tumor growth and Interestingly, we not an of KLF6 by the stress and sodium However, the siRNA knockdown the of KLF6 for ATF3 expression. Thus, we that other mechanisms to KLF6 as that of is to as a tumor suppressor D. S. G. Martignetti J.A. Narla G. Wang R. M.J. Friedman S.L. Cancer Res. 2005; PubMed Scopus Google Scholar). In various protein protein kinase kinase and the KLF6 other mechanisms of of transcription demonstrated a role of ATF3 in tumor and metastasis (14Bandyopadhyay S. Wang Y. Zhan R. Pai S.K. Watabe M. Iiizumi M. Furuta E. Mohinta S. Liu W. Hirota S. Hosobe S. Tsukada T. Miura K. Takano Y. Saito K. Commes T. Piquemal D. Hai T. Watabe K. Cancer Res. 2006; 66: 11983-11990Crossref PubMed Scopus (93) Google as well as in cell A.E. J. P. A. G. W. G. J. 2006; PubMed Scopus Google Scholar). The from be by the dichotomous of ATF3 (15Yin X. Dewille J.W. Hai T. Oncogene. 2008; 27: 2118-2127Crossref PubMed Scopus (160) Google Scholar). Although ATF3 induce apoptosis in cells under conditions, it also growth and metastasis in other cells or under other conditions. that in and apoptosis cancer cell at the enhances apoptosis Oncogene. PubMed Scopus Google Scholar). The KLF6 siRNA that we employed for the knockdown also target the of were shown to the of KLF6 in cell G. Difeo A. Reeves H.L. J. E. Katz A. S. A. S.K. K.E. S.L. A.M. J. S.N. Friedman S.L. Martignetti J.A. Cancer Res. 2005; PubMed Scopus Google Scholar). Interestingly, both and SV2 were to ATF3 and induce apoptosis in PC-3 cells KLF6 suppresses tumor growth by G. Kremer-Tal S. Matsumoto N. Zhao X. Yao S. Kelley K. Tarocchi M. Friedman S.L. Oncogene. 2007; 26: 4428-4434Crossref PubMed Scopus (69) Google these variant still the that are required for activating ATF3 gene whereas the for promoter binding and be through In we identified ATF3 as a key target of KLF6 in tumor ATF3 KLF6-induced apoptosis in stress conditions. in the KLF6 gene can to to apoptosis in prostate cancer cells. We Dr. Tsonwin Hai State for kindly the pCG and pCG-ATF3 expression We also Dr. Shigetaka Kitajima Medical and Dental for the ATF3 promoter reporter plasmids Luc-1850, Luc-632, Luc-111, and with