Title: Regulation of KiSS-1 Metastasis Suppressor Gene Expression in Breast Cancer Cells by Direct Interaction of Transcription Factors Activator Protein-2α and Specificity Protein-1
Abstract: KiSS-1 has been shown to function as a tumor metastasis suppressor gene and reduce the number of metastases in different cancers. The expression of KiSS-1 or KiSS1, like other tumor suppressor, is commonly reduced or completely ablated in a variety of cancers via an unknown mechanism. Here we show that the loss of KiSS-1 expression in highly metastatic breast cancer cell lines correlates directly with the expression levels of two transcription factors, activator protein-2α (AP-2α) and specificity protein 1 (Sp1), which synergistically activate the transcriptional regulation of KiSS-1 in breast cancer cells. Although the KiSS-1 promoter contains multiple AP-2α binding elements, AP-2α-mediated regulation occurs indirectly through Sp1 sites, as determined by deletion and mutation analysis. Overexpression of AP-2α into highly metastatic breast cell lines did not alter KiSS-1 promoter-driven luciferase gene activity. However, co-transfection of AP-2α wild-type or the dominant negative form of AP-2 lacking its C-terminal DNA-binding domain, AP-2B, together with Sp1, increased KiSS-1 promoter activity dramatically, suggesting that AP-2α regulation of KiSS-1 transcription does not require direct binding to the KiSS-1 promoter. Furthermore, we demonstrated that AP-2α directly interacted with Sp1 to form transcription complexes at two tandem Sp1-binding sites of the promoter to activate KiSS-1 transcription. Together, our results indicate that AP-2α and Sp1 are strong transcriptional regulators of KiSS-1 and that loss or decreased expression of AP-2α in breast cancer may account for the loss of tumor metastasis suppressor KiSS-1 expression and thus increased cancer metastasis. KiSS-1 has been shown to function as a tumor metastasis suppressor gene and reduce the number of metastases in different cancers. The expression of KiSS-1 or KiSS1, like other tumor suppressor, is commonly reduced or completely ablated in a variety of cancers via an unknown mechanism. Here we show that the loss of KiSS-1 expression in highly metastatic breast cancer cell lines correlates directly with the expression levels of two transcription factors, activator protein-2α (AP-2α) and specificity protein 1 (Sp1), which synergistically activate the transcriptional regulation of KiSS-1 in breast cancer cells. Although the KiSS-1 promoter contains multiple AP-2α binding elements, AP-2α-mediated regulation occurs indirectly through Sp1 sites, as determined by deletion and mutation analysis. Overexpression of AP-2α into highly metastatic breast cell lines did not alter KiSS-1 promoter-driven luciferase gene activity. However, co-transfection of AP-2α wild-type or the dominant negative form of AP-2 lacking its C-terminal DNA-binding domain, AP-2B, together with Sp1, increased KiSS-1 promoter activity dramatically, suggesting that AP-2α regulation of KiSS-1 transcription does not require direct binding to the KiSS-1 promoter. Furthermore, we demonstrated that AP-2α directly interacted with Sp1 to form transcription complexes at two tandem Sp1-binding sites of the promoter to activate KiSS-1 transcription. Together, our results indicate that AP-2α and Sp1 are strong transcriptional regulators of KiSS-1 and that loss or decreased expression of AP-2α in breast cancer may account for the loss of tumor metastasis suppressor KiSS-1 expression and thus increased cancer metastasis. The vast majority of breast cancer deaths results from complications caused by tumor cell metastasis rather than as a consequence of the original tumor growth. Once tumorigenic cells enter into the vascular and lymphatic systems, they travel to peripheral regions where they invade tissues and form neoplasms. Metastasis is a process requiring detachment of cancer cells from the primary site, survival of sheer forces encountered in the circulation, migration to other organs, attachment to and invasion of tissues, proliferation of these cells at the secondary site, and finally the capacity to enlist neighboring capillaries to supply the tumor with nutrients as it develops (1Pantel K. Brakenhoff R.H. Nat. Rev. Cancer. 2004; 4: 448-456Crossref PubMed Scopus (1063) Google Scholar). Interference at any one of these steps can block this metastatic cascade thereby preventing the formation of metastatic tumor growths. Consequently, there is a growing interest in researching the metastatic process to identify possible ways to inhibit its progression. Metastasis suppressor genes, which inhibit the spread of cancers to secondary sites, have become the target of mounting clinical and basic cancer research. One such gene, KiSS-1 or KiSS1, was originally identified as a metastasis suppressor by microcell-mediated transfer in melanoma lines, by which it was found to reduce tumor cell invasive and migratory properties without affecting their tumorigenicity (2Lee J.H. Welch D.R. Int. J. Cancer. 1997; 71: 1035-1044Crossref PubMed Scopus (161) Google Scholar). Since then, KiSS-1 has been shown to act as a potent anti-metastatic agent either by treatment using synthesized KiSS-1 peptide or upon ectopic expression in highly metastatic cells (2Lee J.H. Welch D.R. Int. J. Cancer. 1997; 71: 1035-1044Crossref PubMed Scopus (161) Google Scholar, 3Masui T. Doi R. Mori T. Toyoda E. Koizumi M. Kami K. Ito D. Peiper S.C. Broach J.R. Oishi S. Niida A. Fujii N. Imamura M. Biochem. Biophys. Res. 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Here, we report that the expression of KiSS-1 metastasis suppressor gene in breast cancer cells is directly correlated with the expression of transcription factors AP-2α and Sp1, and that AP-2α and Sp1 synergistically activate the transcriptional regulation of KiSS-1 in breast cancer cells. Furthermore, we demonstrate that KiSS-1 expression is modulated by AP-2α through direct interaction with the transcription factor Sp1 at two tandem Sp1-binding sites rather than via interaction with the consensus AP-2-binding sites of KiSS-1 promoter. These results offer a mechanism for the loss of KiSS-1 gene expression commonly seen in metastatic breast cancers and provide another molecular mechanism by which AP-2α and Sp1 transcription complex modulates tumorigenesis and tumor progression. Cell Lines, Chemicals, Constructs, and Oligonucleotides—MCF-7, MDA-231, MDA-435, and T47D cells were obtained from the American Type Culture Collection (Manassas, VA). Dulbecco's modified Eagle's medium and RPMI 1640 with phenol red, 100X antibiotics, and fetal bovine serum were purchased from HyClone (Logan, UT). [γ-32P]ATP (300 Ci/mmol) was obtained from PerkinElmer Life Sciences (Wellesley, MA). Poly(dI-dC) and T4 polynucleotide kinase were purchased from Roche Molecular Biochemicals (Indianapolis, IN). Antibodies for Sp1, AP-2α, IgG, and actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Luciferase reagent and lysis buffer were obtained from Promega Corp. (Madison, WI). The 1.2-kb KiSS-1 promoter was cloned from BAC clone RP11–203F10 (accession AL592114) using primers consisting of XhoI and KpnI sites for ligation into the pGL3-basic vector from Promega Corp. (Madison, WI); sense, 5′-GGGGTACCAGACTGCCGGCATGCTT-3′ and antisense, 5′-CCGCTCGAGTTCTCCCCAGCTCCCTGATCACATCC-3′. All other KiSS-1 promoter mutants and truncated fragments were likewise cloned into XhoI and KpnI sites in the pGL3-basic vector. Expression vectors for AP-2α, AP-2B, and Sp1 were cloned as previously described (26Tellez C. Bar-Eli M. Oncogene. 2003; 22: 3130-3137Crossref PubMed Scopus (110) Google Scholar). The Sp1-ΔDBD construct was cloned into pCDNA3.1 (Invitrogen) and was generated from the full-length Sp1 construct with PCR primers, which excluded the DNA-binding region; sense, 5′-CGGAATTCATGAGCGACCAAGATCACTCCATGGATC-3′ and antisense, 5′-CCGCTCGAGGAAGCCATTGCCACTGATATTAAT-3′. Transfection of Breast Cells and Preparation of Nuclear Extracts—Cells were cultured in 6- or 24-well plates in Dulbecco's modified Eagle's medium or RPMI 1640 supplemented with 10% fetal bovine serum. After 18–20 h when cells were ∼60% confluent, reporter gene constructs were transfected using Lipofectamine reagent according to the manufacturer's protocol (Invitrogen). In brief, 1 μg of total DNA was transfected into each well of a 24-well plate using a Lipofectamine to DNA ratio of 2:1 for a period of 6 h. Empty vector was used to offset the difference in DNA concentrations in reactions in which fewer test plasmids were transfected. Transfection reagent was then removed from each well, and cells were incubated in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were harvested after 48 h, and luciferase activity of protein lysates was measured following the manufacturer's protocol (Luciferase Assay System, Promega). To normalize for differences in cell line transfection efficiencies, all cells were transfected with pRSV-β-gal control vector (Promega). β-Galactosidase levels were then measured following the manufacturer's protocol (Galacto-Light Plus, Bedford, MA). For electrophoretic mobility shift assay (EMSA), nuclear extracts from MCF-7 cells were harvested as described previously (38Abdelrahim M. Smith 3rd, R. Burghardt R. Safe S. Cancer Res. 2004; 64: 6740-6749Crossref PubMed Scopus (174) Google Scholar). Concentration of nuclear extracts was determined using BCA assay (Pierce). Aliquots of nuclear protein were frozen and stored at –80 °C until used. Western Immunoblot Analysis—Breast cell lines (2.0 × 107) were seeded in 100-mm Petri dishes with 10 ml of complete medium and incubated overnight. The cells were then scraped off and washed in cold phosphate-buffered saline. The cell pellet was then lysed in 0.5 ml of RIPA buffer (1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 m NaCl, 0.01 m sodium phosphate, pH 7.2, 2 mm EDTA, 50 mm sodium fluoride, 0.2 mm sodium vanadate, 100 units/ml aprotinin). Soluble proteins were then separated by centrifugation at 15,000 × g for 5 min at 4 °C. Protein concentration was determined. Samples were then diluted into loading buffer at 1 mg/ml. Following heat denaturation, samples containing 10 μg of protein were loaded onto and separated on 10% or 15% SDS-PAGE gels as needed. Proteins were then transferred electrophoretically to 0.45-μm nitrocellulose membrane (Pall Corp., Pensacola, FL). After incubating the membranes in blocking solution, primary antibody was added at 1:1,000 dilution, followed by secondary antibody incubation at 1:10,000. Proteins were detected using the SuperSignal West Pico Chemiluminescent substrate (Pierce) according to the manufacturer's instructions. Electrophoretic Mobility Shift Assay—KiSS-1 promoter-derived oligonucleotides were synthesized and annealed, and 5 pmol was 5′-end-labeled using T4 Kinase and [γ32-P]ATP. A 30-μl EMSA reaction containing ∼100 mm potassium chloride, 3 μg of crude nuclear extract, 1 μg of poly(dI-dC) with or without unlabeled competitor oligonucleotide, and 10 fmol of labeled probe was incubated on ice for 20 min. A Sp1-specific antibody was then incubated in appropriate reactions for 20 min on ice. A separate AP-2 antibody was used for gel-shift analysis (Active Motif, Montreal, Canada). DNA·protein complexes were then resolved on 5% PAGE gel at ∼120 V at room temperature for 2 h. Antibody·protein complexes were observed as supershifted or immunodepleted complexes. ChIP Assay—Chromatin immunoprecipitation (ChIP) was performed following the protocol outlined by the ChIP assay kit (Upstate Biotechnology, Lake Placid, NY). Briefly, MCF-7 cells transfected with both AP-2α and Sp1 (2 × 107 cells) were fixed with 1% formaldehyde, scraped into conical tubes, pelleted, and lysed in SDS lysis buffer containing 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, and 1 μg/ml pepstatin A. DNA was sheared to fragments of 200–500 bp by eight 10-s sonications. The chromatin was precleared with salmon sperm DNA/protein A-agarose slurry (Upstate Biotechnology) for 1 h at 4 °C with gentle agitation. The agarose beads were pelleted, and the precleared supernatant was incubated with antibodies to IgG, AP-2α, and Sp1 overnight at 4 °C. The region between –288 and –188 of the KiSS-1 promoter was amplified from the immunoprecipitated chromatin using the following primers: sense, 5′-ATAGCCCATTTCCACGTTG-3′ and antisense, 5′-GGCGGGACTTTCTCCTTC-3′. Following PCR, the 100-bp product was resolved on a 2.5% agarose gel and stained with ethidium bromide. Samples were visualized under UV light. Co-immunoprecipitation Analysis—Binding of AP-2α and Sp1 in transfected MCF-7 cells was examined by immunoprecipitation (IP) and by Western blot analysis. Briefly, cells were lysed with RIPA buffer containing 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 10 mm Tris, pH 7.4, 150 mm NaCl, 1 mm EDTA, 20 mm NaF, 10 mg/ml phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, and 1 mg/ml leupeptin, followed by sonication with a 550 Sonic dismembrator (Fisher Scientific) and immunoprecipitated with the indicated antibodies. Anti-AP-2α and anti-Sp1 immunocomplexes were recovered by using protein A beads (Sigma). All immunoprecipitates were washed four times with lysis buffer and were separated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Millipore). After incubation in TBST buffer (20 mm Tris-HCl, pH 7.5/500 mm NaCl/0.02% Tween 20) containing 0.2% bovine serum albumin and 5% dry milk powder for 2 h, the membranes were probed with the indicated antibodies and visualized with the SuperSignal West Pico detection system (Pierce). Semiquantitative Reverse Transcription-PCR Analysis—Total RNA was isolated from breast cancer cell lines with TRIzol reagent (Invitrogen). First-strand cDNA synthesis was performed using Moloney murine leukemia virus reverse transcriptase and oligo(dT) (Promega) according to the manufacturer's protocol. Primer sequences used for detection of KiSS-1 transcripts were 5′-GCCCACCATGAACTCACTG-3′ and 5′-CTGC-CCCGCACCTGCG-3′. Amplified products were ∼400 bases in length. Additionally, primers for β-actin were 5′-GGCTCCGGCATGTGCAAGGC-3′ and 5′-AGATTTTCTCCATGTCGTCC-3′, which resulted in PCR products of ∼200 bases. Optimal PCR cycles required for linear amplification for each set was determined. β-Actin required 21–23 cycles per reaction, whereas KiSS-1 required 24–28 cycles. PCR products were separated using agarose gels of appropriate concentration, visualized by EtBr staining and quantitated using Alpha Imager software (Alpha Innotech, San Leandro, CA). Endogenous KiSS-1 Expression in Breast Cancer Cell Lines Correlates with AP-2α Expression Levels—We and other laboratories have reported that KiSS-1 expression is lost in highly metastatic breast cancer cells. To determine if loss of the transcription factor AP-2α and Sp1 was directly or inversely associated with loss of KiSS-1, the expression levels of AP-2α and Sp1 levels in both highly metastatic and non-metastatic breast cells were compared with the expression of KiSS-1 (Fig. 1A). Due to the lack of a specific and effective KiSS-1 antibody, KiSS-1 expression level in the breast cell lines was quantitated using RT-PCR and normalized to the β-actin level within each sample. The relatively non-metastatic breast cell line, MCF-7, showed dramatically higher -fold expression as compared with the more metastatic breast tumor cell lines (T47D, MDA-231, and MDA-435). Similarly, Western blot analysis demonstrated that AP-2α expression was lost or barely detectable in the metastatic cell lines, MDA-231 and MDA-435 (Fig. 1A). The expression levels of Sp1 were also examined in these cells. As shown in Fig. 1A