KAI1 Gene Is Engaged in NDRG1 Gene-mediated Metastasis Suppression through the ATF3-NFκB Complex in Human Prostate Cancer

Abstract

NDRG1 and KAI1 belong to metastasis suppressor genes, which impede the dissemination of tumor cells from primary tumors to distant organs. Previously, we identified the metastasis promoting transcription factor, ATF3, as a downstream target of NDRG1. Further analysis revealed that the KAI1 promoter contained a consensus binding motif of ATF3, suggesting a possibility that NDRG1 suppresses metastasis through inhibition of ATF3 expression followed by activation of the KAI1 gene. In this report, we found that ectopic expression of NDRG1 was able to augment endogenous KAI1 gene expression in prostate cancer cell lines, whereas silencing NDRG1 was accompanied with significant decrease in KAI1 expression in vitro and in vivo. In addition, our results of ChIP analysis indicate that ATF3 indeed bound to the promoter of the KAI1 gene. Importantly, our promoter-based analysis revealed that ATF3 modulated KAI1 transcription through cooperation with other endogenous transcription factor as co-activator (ATF3-JunB) or co-repressor (ATF3-NFκB). Moreover, loss of KAI1 expression significantly abrogated NDRG1-mediated metastatic suppression in vitro as well as in a spontaneous metastasis animal model, indicating that KA11 is a functional downstream target of the NDRG1 pathway. Our result of immunohistochemical analysis showed that loss of NDRG1 and KAI1 occurs in parallel as prostate cancer progresses. We also found that a combined expression status of these two genes serves as a strong independent prognostic marker to predict metastasis-free survival of prostate cancer patients. Taken together, our result revealed a novel regulatory network of two metastasis suppressor genes, NDRG1 and KAI1, which together concerted metastasis-suppressive activities through an intrinsic transcriptional cascade. NDRG1 and KAI1 belong to metastasis suppressor genes, which impede the dissemination of tumor cells from primary tumors to distant organs. Previously, we identified the metastasis promoting transcription factor, ATF3, as a downstream target of NDRG1. Further analysis revealed that the KAI1 promoter contained a consensus binding motif of ATF3, suggesting a possibility that NDRG1 suppresses metastasis through inhibition of ATF3 expression followed by activation of the KAI1 gene. In this report, we found that ectopic expression of NDRG1 was able to augment endogenous KAI1 gene expression in prostate cancer cell lines, whereas silencing NDRG1 was accompanied with significant decrease in KAI1 expression in vitro and in vivo. In addition, our results of ChIP analysis indicate that ATF3 indeed bound to the promoter of the KAI1 gene. Importantly, our promoter-based analysis revealed that ATF3 modulated KAI1 transcription through cooperation with other endogenous transcription factor as co-activator (ATF3-JunB) or co-repressor (ATF3-NFκB). Moreover, loss of KAI1 expression significantly abrogated NDRG1-mediated metastatic suppression in vitro as well as in a spontaneous metastasis animal model, indicating that KA11 is a functional downstream target of the NDRG1 pathway. Our result of immunohistochemical analysis showed that loss of NDRG1 and KAI1 occurs in parallel as prostate cancer progresses. We also found that a combined expression status of these two genes serves as a strong independent prognostic marker to predict metastasis-free survival of prostate cancer patients. Taken together, our result revealed a novel regulatory network of two metastasis suppressor genes, NDRG1 and KAI1, which together concerted metastasis-suppressive activities through an intrinsic transcriptional cascade. Although significant advances have been made in reducing mortality rates, the majority of cancer patients are still diagnosed at an advanced stage and ultimately die from sequelae of metastatic disease. Metastasis involves a complex process through which malignant cancer cells leave a primary organ site, journey to a distant site via circulation, and finally establish a clinically detectable mass in a distant organ, and therefore, the metastatic progression requires dysregulation of a series of genes and related signaling. Metastasis suppressor genes are negative regulators of metastasis, which inhibit metastasis but do not affect the ability of the transformed cells to generate a tumor at the primary site (1Berger J.C. Vander Griend D.J. Robinson V.L. Hickson J.A. Rinker-Schaeffer C.W. Cancer Biol. Ther. 2005; 4: 805-812Crossref PubMed Scopus (47) Google Scholar, 2Nash K.T. Welch D.R. Front. Biosci. 2006; 11: 647-659Crossref PubMed Scopus (75) Google Scholar, 3Shevde L.A. Welch D.R. 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Lancet. 2007; 369: 1742-1757Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar). However, the detailed molecular mechanism of how these genes are regulated and their functions are less elucidated. KAI1, also known as CD82, was discovered initially from T-cell activation study and was identified later as a prostate-specific tumor metastasis suppressor gene (8Gaugitsch H.W. Hofer E. Huber N.E. Schnabl E. Baumruker T. Eur. J. Immunol. 1991; 21: 377-383Crossref PubMed Scopus (87) Google Scholar, 9Ichikawa T. Ichikawa Y. Isaacs J.T. Cancer Res. 1991; 51: 3788-3792PubMed Google Scholar, 10Ichikawa T. Ichikawa Y. Dong J. Hawkins A.L. Griffin C.A. Isaacs W.B. Oshimura M. Barrett J.C. Isaacs J.T. Cancer Res. 1992; 52: 3486-3490PubMed Google Scholar, 11Dong J.T. Lamb P.W. Rinker-Schaeffer C.W. Vukanovic J. Ichikawa T. Isaacs J.T. Barrett J.C. Science. 1995; 268: 884-886Crossref PubMed Scopus (764) Google Scholar). It is ubiquitously expressed in normal tissues with high mRNA levels in spleen, placenta, kidney, and prostate, whereas decrease or loss of its expression is constantly found in the clinically advanced cancers (12Liu W.M. Zhang X.A. Cancer Lett. 2006; 240: 183-194Crossref PubMed Scopus (116) Google Scholar). Consistently, inverse correlations between KAI1 expression and the invasive and metastatic potential as well as poor survival of patients have been observed frequently in a wide range of malignancies (12Liu W.M. Zhang X.A. Cancer Lett. 2006; 240: 183-194Crossref PubMed Scopus (116) Google Scholar). KAI1 belongs to the tetraspanin transmembrane protein superfamily, and it is found to be associated with other tetraspanins (CD9 and CD81), integrins (β1 and β2), immunoreceptors (MHCI and II, EWI1/PGRL, CD4, CD8, CD19, and CD46), growth factors and receptors (EGF and EGFR), as well as intracellular signaling proteins (PKC) (12Liu W.M. Zhang X.A. Cancer Lett. 2006; 240: 183-194Crossref PubMed Scopus (116) Google Scholar). KAI1 was found previously to inhibit cell motility by regulating the biological activities of its associated proteins and/or reorganizing plasma membrane microdomains (12Liu W.M. Zhang X.A. Cancer Lett. 2006; 240: 183-194Crossref PubMed Scopus (116) Google Scholar). This process occasionally induces apoptosis by releasing intracellular glutathione and accumulating intracellular reactive oxygen intermediates (13Ono M. Handa K. Withers D.A. Hakomori S.I. Cancer Res. 1999; 59: 2335-2339PubMed Google Scholar, 14Schoenfeld N. Bauer M.K. Grimm S. FASEB J. 2004; 18: 158-160Crossref PubMed Scopus (35) Google Scholar). Moreover, we have demonstrated recently that KAI1 exerts its metastasis suppressor function by directly binding to Duffy blood group, chemokine receptor on endothelial cells, thereby inducing senescence signaling in tumor cells in circulation (15Bandyopadhyay 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). Because mutations and loss of heterozygosity of the KAI1 gene is rare, the down-regulation of this gene is not likely because of genetic alterations but is rather due to modulation of transcriptional and post-transcriptional regulation (16Dong J.T. Suzuki H. Pin S.S. Bova G.S. Schalken J.A. Isaacs W.B. Barrett J.C. Isaacs J.T. Cancer Res. 1996; 56: 4387-4390PubMed Google Scholar, 17Kawana Y. Komiya A. Ueda T. Nihei N. Kuramochi H. Suzuki H. Yatani R. Imai T. Dong J.T. Imai T. Yoshie O. Barrett J.C. Isaacs J.T. Shimazaki J. Ito H. Ichikawa T. Prostate. 1997; 32: 205-213Crossref PubMed Scopus (63) Google Scholar, 18Tagawa K. Arihiro K. Takeshima Y. Hiyama E. Yamasaki M. Inai K. Jpn. J. Cancer Res. 1999; 90: 970-976Crossref PubMed Scopus (38) Google Scholar, 19Jackson P. 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Other molecules involved in KAI1 transcriptional regulation include NFκB, β-catenin/Reptin, Tip60/Fe65, N-CoR/TBL2/HDAC3 and AP-1 (23Telese F. Bruni P. Donizetti A. Gianni D. D'Ambrosio C. Scaloni A. Zambrano N. Rosenfeld M.G. Russo T. EMBO Rep. 2005; 6: 77-82Crossref PubMed Scopus (82) Google Scholar, 24Baek S.H. Ohgi K.A. Rose D.W. Koo E.H. Glass C.K. Rosenfeld M.G. Cell. 2002; 110: 55-67Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 25Kim J.H. Kim B. Cai L. Choi H.J. Ohgi K.A. Tran C. Chen C. Chung C.H. Huber O. Rose D.W. Sawyers C.L. Rosenfeld M.G. Baek S.H. Nature. 2005; 434: 921-926Crossref PubMed Scopus (261) Google Scholar, 26Dong J.T. Isaacs W.B. Barrett J.C. Isaacs J.T. Genomics. 1997; 41: 25-32Crossref PubMed Scopus (71) Google Scholar, 27Marreiros A. Dudgeon K. Dao V. Grimm M.O. Czolij R. Crossley M. Jackson P. Oncogene. 2005; 24: 637-649Crossref PubMed Scopus (69) Google Scholar, 28Marreiros A. Czolij R. Yardley G. Crossley M. Jackson P. Gene. 2003; 302: 155-164Crossref PubMed Scopus (34) Google Scholar). Interestingly, these transcription factors were frequently found to coordinately regulate the expression of KAI1 as either co-activator or co-repressor. Therefore, studies on these transcription factors may aid in elucidating the mechanism leading to KAI1 suppression and following metastatic progression. NDRG1 (N-Myc downstream regulated gene 1) was originally isolated as a novel gene that was induced strongly during differentiation of colon carcinoma cell lines (29van Belzen N. Dinjens W.N. Diesveld M.P. Groen N.A. van der Made A.C. Nozawa Y. Vlietstra R. Trapman J. Bosman F.T. Lab. Invest. 1997; 77: 85-92PubMed Google Scholar). Recent studies demonstrated that the NDRG1 gene is controlled by a variety of factors and stimuli related to cancer progression, including oncogenes, tumor suppressors, hypoxic microenvironment, and hormone dysregulation (30Kovacevic Z. Richardson D.R. Carcinogenesis. 2006; 27: 2355-2366Crossref PubMed Scopus (159) Google Scholar, 31Ellen T.P. Ke Q. Zhang P. Costa M. Carcinogenesis. 2008; 29: 2-8Crossref PubMed Scopus (167) Google Scholar). Clinical studies also provided compelling evidence that reduced expression of the NDRG1 gene was significantly associated with poor overall survival rate in pancreatic ductal adenocarcinoma, glioma, prostate, breast, and colorectal cancers (32Iiizumi M. Liu W. Pai S.K. Furuta E. Watabe K. Biochim. Biophys. Acta. 2008; 1786: 87-104PubMed Google Scholar). The significant inverse correlation of NDRG1 expression with the extent of metastasis in a clinical setting raised an important question as to whether the down-regulation of NDRG1 is cause or result of metastases. To address this issue, we overexpressed the NDRG1 gene in a highly metastatic prostate cell line AT6.1 and implanted it into severe combined immunodeficiency mice (33Bandyopadhyay S. Pai S.K. Gross S.C. Hirota S. Hosobe S. Miura K. Saito K. Commes T. Hayashi S. Watabe M. Watabe K. Cancer Res. 2003; 63: 1731-1736PubMed Google Scholar). Our results indicate that NDRG1 has the ability to suppress the metastatic process of prostate cancer cells without affecting tumorigenicity in vivo. Similar metastasis suppressor effect of NDRG1 was also observed in other animal models including colon, bladder, and pancreatic carcinoma cells (34Guan R.J. Ford H.L. Fu Y. Li Y. Shaw L.M. Pardee A.B. Cancer Res. 2000; 60: 749-755PubMed Google Scholar, 35Kurdistani S.K. Arizti P. Reimer C.L. Sugrue M.M. Aaronson S.A. Lee S.W. Cancer Res. 1998; 58: 4439-4444PubMed Google Scholar, 36Maruyama Y. Ono M. Kawahara A. Yokoyama T. Basaki Y. Kage M. Aoyagi S. Kinoshita H. Kuwano M. Cancer Res. 2006; 66: 6233-6242Crossref PubMed Scopus (140) Google Scholar). Furthermore, NDRG1 was shown to suppress the invasive and angiogenic abilities of aggressive cancer cells (36Maruyama Y. Ono M. Kawahara A. Yokoyama T. Basaki Y. Kage M. Aoyagi S. Kinoshita H. Kuwano M. Cancer Res. 2006; 66: 6233-6242Crossref PubMed Scopus (140) Google Scholar, 37Nishio S. Ushijima K. Tsuda N. Takemoto S. Kawano K. Yamaguchi T. Nishida N. Kakuma T. Tsuda H. Kasamatsu T. Sasajima Y. Kage M. Kuwano M. Kamura T. Cancer Lett. 2008; 264: 36-43Crossref PubMed Scopus (66) Google Scholar, 38Hosoi F. Izumi H. Kawahara A. Murakami Y. Kinoshita H. Kage M. Nishio K. Kohno K. Kuwano M. Ono M. Cancer Res. 2009; 69: 4983-4991Crossref PubMed Scopus (84) Google Scholar). A recent study employing whole genome gene array analysis in examining the functions of NDRG1 in a number of different cancer cells strongly indicate the pleiotropic nature of NDRG1 in suppressing metastasis (39Xu X. Sutak R. Richardson D.R. Mol. Pharmacol. 2008; 73: 833-844Crossref PubMed Scopus (45) Google Scholar). We previously performed the Affymetrix gene array analysis and found that NDRG1 suppressed metastasis of prostate tumor cells by inhibiting the transcription factor ATF3 (15Bandyopadhyay 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). Consistent with our observation, Ishiguro et al. (40Ishiguro T. Nakajima M. Naito M. Muto T. Tsuruo T. Cancer Res. 1996; 56: 875-879PubMed Google Scholar) showed that ectopic expression of ATF3 converted the low metastatic potential melanoma cell line to become highly metastatic. Moreover, ATF3 expression appears to be required for the maintenance of a high metastatic state of melanoma and colon cancer cells (41Ishiguro T. Nagawa H. Naito M. Tsuruo T. Jpn. J. Cancer Res. 2000; 91: 833-836Crossref PubMed Scopus (44) Google Scholar). ATF3 is a member of cAMP-responsive element binding protein (ATF/CREB) family of basic leucine zipper transcription factors (42Hai T. Wolfgang C.D. Marsee D.K. Allen A.E. Sivaprasad U. Gene Expr. 1999; 7: 321-335PubMed Google Scholar). Emerging evidence suggests that ATF3 plays a critical role in metastatic progression in a cell context-dependent manner. To gain further mechanistic insight into the functional role of NDRG1, we sought to identify and characterize the possible downstream targets of ATF3 that are involved in tumor metastasis. The result of our bioinformatic analysis for the promoters of metastasis-related genes revealed that there were a number of genes whose promoter contained the ATF3-responsive consensus sequence, TGACGTCA. Among these, we identified proapoptotic gene GADD153/CHOP10, cell adhesion molecular E-selectin, tumor suppressor p53, and metastasis suppressor KAI1 as potential targets of ATF3. These clues prompted us to examine a possible link between two metastasis suppressor genes, NDRG1 and KAI1, through ATF3 in the current study. Human prostate cancer cell line PC3mm and rat prostatic carcinoma cell line AT6.1 were kindly provided by Drs. I. J. Fidler (The University of Texas MD Anderson Cancer Center, Houston, TX) and C. Rinker-Schaeffer (University of Chicago), respectively. The PC3mm/Tet 2The abbreviations used are: Tet, tetracycline; TRAMP, transgenic adenocarcinoma of the mouse prostate. cell line was established previously as a derivative of PC3mm and contains a tetracycline-inducible suppressor. The human prostate cancer cell line DU145, prostate epithelial cell line RWPE1, and colon cancer cell line HT38 were obtained from the American Type Culture Collection (Manassas, VA). NDRG1- and KAI1-overexpressing stable clones of AT6.1 cells were established as described previously (33Bandyopadhyay S. Pai S.K. Gross S.C. Hirota S. Hosobe S. Miura K. Saito K. Commes T. Hayashi S. Watabe M. Watabe K. Cancer Res. 2003; 63: 1731-1736PubMed Google Scholar). For AT6.1/NDRG1/shKAI1, shRNA for KAI1 (Open Biosystems) or the vector alone was transfected into AT6.1/NDRG1 cells, and the puromycin-resistant clones were selected. The expression of KAI1 in the cloned cells was tested by RT-PCR. All cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, streptomycin (100 μg/ml), penicillin (100 units/ml), and 250 nm dexamethasone (Sigma) and grown at 37 °C in a 5% CO2 atmosphere. For all transfection experiments, Lipofectamine 2000 (Invitrogen) was used according to the manufacturer's protocol. The luciferase activities were measured by using the Dual-Luciferase Reporter Assay System (Promega, Madison, MI) and a luminometer (Berthold Detection Systems, Huntsville, Alabama). For each transfection experiment, the Renilla expression plasmid phRG-TK (Promega) was co-transfected as an internal control, and the promoter activities were normalized accordingly. Immunohistochemical analysis on paraffin-embedded, surgically resected specimens of prostate and breast was carried out using anti-NDRG1 rabbit polyclonal antibody (from Dr. Commes) and anti-KAI1 antibody (kindly provided by Dr. Yoshie). Briefly, sections were deparaffinized, rehydrated, and heated at 80 °C for 20 min in 25 mm sodium citrate buffer (pH 9) for antigen exposure. They were then treated with 3% H2O2 to block endogenous peroxidase activity and further incubated with primary antibody for 1 h at 24 °C. After washing with Tris-buffered saline/0.1% Tween 20, the sections were incubated with horseradish peroxidase-conjugated rabbit-specific IgG (Dako). The sections were washed extensively, and 3,3-diaminobenzidine substrate chromogen solution was applied followed by counterstaining with hematoxylin. The GEO database (accession no. GSE21034, n = 218) was used to evaluate the clinical relevance of NDRG1 and KAI1 in prostate cancer. The data were log2-transformed, with the median set as zero and with S.D. set as one. Each patient was assigned to have positive or negative expression of each gene and was matched with metastasis-free survival. The gene expressions in normal, primary tumor, or metastatic sites in patients who have either metastatic or nonmetastatic disease were compared using Box-and-whisker plot analysis and evaluated by the Mann-Whitney test. The association between genes and clinical outcomes was calculated by the Pearson χ2 test. The Kaplan-Meier method was used to calculate the overall survival rate, and prognostic significance was evaluated by the log-rank test. Multivariate analysis for the prognostic value of gene signatures was performed by the Cox proportional hazard regression model. For all statistical analysis, Prism and SPSS software were used. For in vitro experiments and animal studies, results are reported as mean ± S.D. (or mean ± S.E.) as indicated in the figure legends. Statistical significance was determined by a two-sided Student's test. 4–6-Week-old severe combined immunodeficiency mice (Harlan Sprague-Dawley, Indianapolis, IN) were used for the spontaneous metastasis studies. 0.5 × 106 cells in 0.1 ml of PBS were injected subcutaneously into the dorsal flank of mice. Mice were monitored daily, and the tumor volume was measured as an index of the growth rate and calculated as (width + length)/2 × width × length × 0.5236. Mice were sacrificed 30 days after the inoculation of cells, and metastatic lesions were counted macroscopically. We first examined the effect of NDRG1 on the expression of the KAI1 gene in two human prostate cancer cell lines, DU145 and PC3mm/Tet-FLAG-NDRG1 (referred to as PC3mm/Tet), by quantitative RT-PCR and Western blot. NDRG1 expression was induced by tetracycline treatment in PC3mm-Tet cells. Alternatively, the NDRG1 expression plasmid was transfected into DU145 cells. We found that the expression of KAI1 gene was significantly up-regulated by ectopic expression of NDRG1 (Fig. 1A). To further examine whether NDRG1 has an effect on the transcription of the KAI1 gene, we transfected the KAI1 reporter plasmid into PC3mm/Tet and DU145 cells, which were then forced to overexpress the NDRG1 gene. As shown in Fig. 1B, our results showed that NDRG1 expression indeed significantly augmented KAI1 gene transcription. Moreover, when PC3mm cells were treated with Dp44mT, an NDRG1-specific agonist, the expression of the KAI1 gene was also significantly elevated in a dose-dependent manner (Fig. 1C, lower panel). The increased KAI1 expression in the Dp44mT-treated cells was also confirmed by immunofluorescence staining (Fig. 1C, upper panel). Furthermore, we introduced NDRG1 shRNA into nonmalignant prostate epithelial cell RWPE1 or NDRG1 siRNA into colon carcinoma cell HT38 because both cell lines are known to express the KAI1 gene. We found that the specific knockdown of NDRG1 indeed significantly down-regulated the expression of the KAI1 gene in these cell lines (Fig. 1D). We also examined the expression of KAI1 and NDRG1 in a panel of prostate and breast cancer cell lines (n = 11 and 39 for prostate and breast cancer cell lines, respectively). We found that KAI1 mRNA expression was significantly correlated with NDRG1 expression in multiple cancer cell lines (p = 0.0014 and 0.0004 for prostate (upper panel) and breast (lower panel) cancer cell lines, respectively, Fig. 1E), indicating that the expression of NDRG1 and KAI1 generally has a positive correlation in human prostate and breast cancer cells. To further examine the relevance of NDRG1 and KAI1 in vivo, we compared the expression of Ndrg1 and Kai1 in NDRG1 knock-out (Ndrg1−/−) and syngenic wild type mice (Ndrg1+/+). Consistent with the results of our in vitro studies, we found that Kai1 mRNA expressed at a significantly lower level in Ndrg1 knock-out mice compared with that in wild type mice (Fig. 1F). To identify the NDRG1-responsive sequence on the KAI1 promoter, we generated a series of luciferase reporter plasmids containing up to −2900, −1118, −730, and −332 bases of the KAI1 promoter, and luciferase reporter activities were measured in PC3mm/Tet cells with or without induction of NDRG1 expression. As shown in Fig. 2A, the deletion between −1118 and −730 bases of the KAI1 promoter region almost completely abolished the ability of this promoter to respond to NDRG1. We also examined the KAI1 promoter activity in DU145 cells, which was transfected with the NDRG1 expression plasmid and observed a similar result. These results suggest that the region between −1118 and −730 contains an NDRG1-responsive sequence. When we examined the sequence of this region, we found three potential binding sites for ATF3, and one of these was a complete match to the consensus sequence. To further determine whether ATF3 binds to the KAI1 promoter, ChIP assay was performed, and the results clearly showed that ATF3 indeed bound to the predicted region of the KAI1 promoter, whereas induction of NDRG1 expression significantly blocked this binding (Fig. 2A, lower panel). Consistently, we found that silencing the expression of ATF3 by siRNA in PC3mm cells resulted in significant up-regulation of KAI1 mRNA expression (Fig. 2B). Therefore, our results strongly suggest that NDRG1 regulates KAI1 expression by modulating ATF3 signaling pathway. Surprisingly, however, we found that ectopic expression of ATF3 in PC3mm cells significantly up-regulated KAI1 promoter activity and that the mutation of the ATF3-binding consensus sequence on the KAI1 promoter abolished this responsiveness to ATF3 (Fig. 2C, left panel). ATF3 is known to be a bidirectional transcription factor, which either activates or represses transcription dependent on its dimerized partner in a cell context-dependent manner (42Hai T. Wolfgang C.D. Marsee D.K. Allen A.E. Sivaprasad U. Gene Expr. 1999; 7: 321-335PubMed Google Scholar). Interestingly, the NFκB subunit p50 was reported previously to be involved in a protein complex that was capable of inhibiting KAI1 transcription (25Kim J.H. Kim B. Cai L. Choi H.J. Ohgi K.A. Tran C. Chen C. Chung C.H. Huber O. Rose D.W. Sawyers C.L. Rosenfeld M.G. Baek S.H. Nature. 2005; 434: 921-926Crossref PubMed Scopus (261) Google Scholar), and p50 is also known to be able to form a dimeric complex with ATF family members (53Gilchrist M. Thorsson V. Li B. Rust A.G. Korb M. Roach J.C. Kennedy K. Hai T. Bolouri H. Aderem A. Nature. 2006; 441: 173-178Crossref PubMed Scopus (629) Google Scholar). Therefore, we examined a possibility that ATF3 coordinates with p50 to repress KAI1 transcription. We found that ATF3 was indeed pulled down with p50 (Fig. 2C, right panel) and works together to suppress KAI1 promoter activity after co-transfection of ATF3 and p50 expression plasmids in PC3mm cells (Fig. 2C, left panel). However, ATF3 and p50 failed to down-regulate KAI1 promoter activity when ATF3 binding sites were mutated (Fig. 2C, left panel), suggesting that p50 cooperates with ATF3 to suppress KAI1 expression. To further verify this result, we examined an effect of the p50 inhibitor, pyrrolidine dithiocarbamate, on the expression of the KAI1 gene in the presence or absence of NDRG1. As shown in Fig. 2D, either induction of NDRG1 or inhibition of p50 significantly up-regulated KAI1 transcriptional activity in the PC3mm cell, and a combination of NDRG1 and pyrrolidine dithiocarbamate further up-regulated KAI1 promoter activity. However, neither of the treatments induced suppression of KAI1 promoter activity when the ATF3 consensus binding site was mutated. These results strongly support our notion that NDRG1 up-regulates KAI1 by blocking the suppressor activity of the ATF3-p50 complex on the KAI1 promoter. Notably, it was reported previously that p50 dimerized and bound to a region between −6631 and −6996 bp upstream of the KAI1 gene transcription start site (23Telese F. Bruni P. Donizetti A. Gianni D. D'Ambrosio C. Scaloni A. Zambrano N. Rosenfeld M.G. Russo T. EMBO Rep. 2005; 6: 77-82Crossref PubMed Scopus (82) Google Scholar, 24Baek S.H. Ohgi K.A. Rose D.W. Koo E.H. Glass C.K. Rosenfeld M.G. Cell. 2002; 110: 55-67Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar). To further corroborate that p50 is involved in ATF3-mediated KAI1 transcriptional regulation, we performed a ChIP assay by precipitating the p50-chromatin complex followed by quantitative PCR analysis with the pairs of primers specific to the region of ATF3 binding site. We found that p50 indeed bound to the proximal region of ATF3 binding site on the KAI1 promoter by forming a complex with ATF3, which was negatively regulated by NDRG1 (Fig. 2E). ATF3 can form a complex with various other members in the ATF family such as ATF2 and ATF4, as well as AP-1 proteins (27Marreiros A. Dudgeon K. Dao V. Grimm M.O. Czolij R. Crossley M. Jackson P. Oncogene. 2005; 24: 637-649Crossref PubMed Scopus (69) Google Scholar, 28Marreiros A. Czolij R. Yardley G. Crossley M. Jackson P. Gene. 2003; 302: 155-164Crossref PubMed Scopus (34) Google Scholar). Interestingly, we found that ATF3, in combination with the AP-1 family protein JunB, significantly enhanced KAI1 transcriptional activity compared with each alone (Fig. 2F). Taken together, our result indicates that ATF3 modulates KAI1 expression through an intrinsic mechanism that is dependent on the dynamic cellular context of tumor progression. The positive regulatory role of NDRG1 in KAI1 expression prompted us to examine the functional relevance of these two metastasis suppressors in tumor progression. We established permanent cell lines expressing NDRG1 with or without knocking down the KAI1 gene using the highly metastatic prostate cancer cell line AT6.1 (Fig. 3, A and B). These cell lines were then examined for their metastatic behavior through a series of functional assays. As we have reported previously, the expression of NDRG1 sign