Activator Protein-2 Overexpression Accounts for Increased Insulin

Research Article

Activator Protein-2 Overexpression Accounts for Increased Insulin Receptor Expression in Human Breast Cancer Francesco Paonessa,1 Daniela Foti,1 Vanessa Costa,1 Eusebio Chiefari,1 Giuseppe Brunetti,3 Francesco Leone,2 Francesco Luciano,1 Frank Wu,4 Amy S. Lee,4 Elio Gulletta,1 Alfredo Fusco,5 and Antonio Brunetti1

1Dipartimento di Medicina Sperimentale e Clinica G. Salvatore, Universita`di Catanzaro Magna Græcia; 2Unita`Operativa di Chirurgia Generale Azienda Ospedaliera Pugliese-Ciaccio, Catanzaro, Italy; 3Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita` di Milano, Milan, Italy; 4Department of Biochemistry and Molecular Biology and the USC Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California; and 5Dipartimento di Biologia e Patologia Cellulare e Molecolare c/o Istituto di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Universita`di Napoli Federico II, e NOGEC-CEINGE, Biotecnologie Avanzate, Napoli, Italy

Abstract insulin-binding domain and two transmembrane h-subunits that have ligand-activated tyrosine kinase activity (1–3). When insulin Various studies have shown that the insulin receptor (IR) is binds to the IR, the receptor is first activated by tyrosine increased in most human breast cancers, and both ligand- autophosphorylation, and then the IR tyrosine kinase phosphor- dependent malignant transformation and increased cell ylates various effector molecules, such as IRS-1, leading to growth occur in cultured breast cells overexpressing the IR. hormone action (1–3). Overexpression of receptor molecules with However, although numerous in vivo and in vitro observations intrinsic tyrosine kinase activity in cells has been involved in the have indicated an important contributory role for the IR in induction of a transformed phenotype (4, 5), and the mediation of a breast cancer cell biology, the molecular mechanisms ac- proliferative response through the IR has been widely shown in counting for increased IR expression in breast tumors have various transformed cells (6–8). In epithelial cells, the IR is usually not previously been elucidated. Herein, we did immunoblot expressed at low levels. However, it has been shown experimentally analyses of nuclear protein from cultured breast cancer cells that overexpression of IRs in these cells can increase responses to and normal and tumoral tissues from breast cancer patients insulin and induce a ligand-mediated neoplastic transformation. In combined with promoter studies by using a series of human this regard, it has been shown that overexpression of functional IRs wild-type and mutant IR promoter constructs. We provide can occur in human breast cancer and other epithelial tumors, evidence that IR overexpression in breast cancer is dependent including ovarian and colon cancer, in which the IR may exert on the assembly of a transcriptionally active multiprotein- its oncogenic potential by directly affecting cell metabolism and/or DNA complex, which includes the high-mobility group A1 by synergizing with other oncogenes in influencing growth and (HMGA1) protein, the developmentally regulated activator differentiation (6–10). However, very little is known about the protein-2 (AP-2) transcription factor and the ubiquitously molecular mechanisms that account for the increased IR expres- expressed transcription factor Sp1. In cultured breast cancer sion in human breast cancer, and understanding these mechanisms cells and human breast cancer specimens, the expression of can be of valuable help from both biological and clinical aspects. AP-2 was significantly higher than that observed in cells and As part of an investigation into the molecular basis of regulation tissues derived from normal breast, and this overexpression of the human IR gene, we previously showed that IR gene paralleled the increase in IR expression. However, AP-2 DNA- IR expression in eukaryotic cells is positively regulated by the binding activity was undetectable with the gene promoter, architectural factor high-mobility group A1 (HMGA1; refs. 11–13), suggesting that transactivation of this gene by AP-2 might a small basic protein that binds to AT-rich regions of certain gene occur indirectly through physical and functional cooperation promoters and functions mainly as a specific cofactor for gene with HMGA1 and Sp1. Our findings support this hypothesis activation (14–17). HMGA1 by itself has no intrinsic transcriptional and suggest that in affected individuals, hyperactivation of the activity; rather, it has been shown to trans-activate promoters AP-2 gene through the overexpression of IR may play a key through mechanisms that facilitate the assembly and stability of role in breast carcinogenesis. (Cancer Res 2006; 66(10): 5085-93) stereospecific DNA-protein complexes that drive gene transcription (14–17). HMGA1 performs this task by modifying DNA conforma- Introduction tion and by recruiting transcription factors to the transcription The peptide hormone insulin exerts its biological effects by start site, facilitating DNA-protein and protein-protein interactions interacting with the insulin receptor (IR), a specific glycoprotein in (14–17). We showed that transcriptional activation of the human the plasma membrane of insulin target cells, which generates IR promoter requires the assembly of a transcriptionally active intracellular signals via a tyrosine kinase cascade (1). The IR multiprotein-DNA complex, which includes, in addition to HMGA1, belongs to the tyrosine kinase growth factor receptor family and the ubiquitously expressed transcription factor Sp1 and the consists of two identical extracellular a-subunits that contain the CCAAT-enhancer binding protein h (C/EBPh; ref. 12). By physically interacting with Sp1 and C/EBPh, HMGA1 facilitates the binding of both factors to the IR promoter in vitro and supports trans- Requests for reprints: Antonio Brunetti, Cattedra di Endocrinologia, Policlinico criptional synergism between these factors in vivo (12). Mater Domini, Universita` di Catanzaro Magna Græcia, Via T. Campanella 115, The transcription factor activator protein-2 (AP-2) activates Catanzaro, Italy. Phone: 39-961-712257; Fax: 39-961-775373; E-mail: [email protected]. I2006 American Association for Cancer Research. transcription via GC-rich DNA sequences (18, 19), and its doi:10.1158/0008-5472.CAN-05-3678 expression is required for normal growth and morphogenesis www.aacrjournals.org 5085 Cancer Res 2006; 66: (10). May 15, 2006

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2006 American Association for Cancer Research. Cancer Research during mammalian development (20). An involvement of AP-2 1:500), anti-AP-2a (H-79; 1:1,000), and anti-IRh (C-19; 1:1,000; Santa Cruz family of proteins in the etiology of human breast cancer has been Biotechnology, Santa Cruz, CA). For covalent coupling of antibodies to shown in previous studies, in which a significant up-regulation of protein A-Sepharose (Amersham, Piscataway, NJ), anti-HMGA1 or anti-Sp1 AP-2a in breast cancer specimens has been observed (21). In polyclonal antibody was mixed with beads and bound for 1 hour with rotation at room temperature. After extensive washing with 200 mmol/L addition, AP-2 genes are expressed in many human breast cancer sodium borate (pH 8), solid dimethyl pimelimidate (Sigma, St. Louis, MO) cell lines, and AP-2 consensus binding sites can be detected in both was added to a final concentration of 20 mmol/L, and the components were c-erbB-2 and estrogen receptor promoters, whose activity is mixed on roller for 30 minutes at room temperature. To stop the reaction, increased in breast cancers (22, 23). In our study, overexpression the beads were washed twice in 200 mmol/L ethanolamine (pH 8) and of the IR in both human breast cancer cell lines and in whole breast incubated on roller for 2 hours at room temperature in 200 mmol/L tumor tissue correlated closely with AP-2a mRNA and protein ethanolamine. Antibody-coupled protein A beads were washed twice in PBS expression levels. Therefore, to explore the biochemical mecha- solution and used in immunoprecipitation studies. 35 nisms involved in IR overexpression in breast cancer, we have Glutathione S-transferase pull-down assay. S-labeled AP-2 and its in vitro examined the functional significance of AP-2a for IR gene trans- deletion mutants were synthesized by using the TNT-T7 quick- cription using the human AP-2-deficient HepG2 cell line. There are coupled transcription/translation system (Promega, Madison, WI). Gluta- thione S-transferase (GST) fusion protein expression vectors for HMGI and no AP-2 DNA-binding sites on the promoter region of the IR gene; derivatives (kindly provided by Dimitris Thanos), Sp1 (a kind gift from Hans however, overexpression of AP-2 in HepG2 cells greatly enhanced IR Rotheneder and Erhard Wintersberger), and the pGEX-2TK control vector gene transcription, supporting a role for this transcription factor (Amersham) were transformed into the BL21 strain of Escherichia coli in the regulation of IR gene expression. In this article, we show for (Stratagene, La Jolla, CA), expanded in suspension culture, and induced the first time that AP-2 physically interacts with HMGA1 and Sp1 for 2 hours with 0.5 mmol/L isopropyl-D-thiogalactopyranoside (Sigma). in vitro and in vivo, and physical interaction between these Bacteria were pelleted, sonicated in ice-cold PBS lysis buffer [1% NP40; 10% factors contributes to efficient functional cooperation in the trans- glycerol; 1 mmol/L DTT; 1 mmol/L phenylmethylsulfonyl fluoride (PMSF); activation of the IR gene by AP-2. Our findings show that AP-2a 10 Ag of pepstatin A, leupeptin, and aprotinin/mL; and 0.4 mg of lysozyme/ may play significant molecular roles in IR overexpression in malig- mL], and centrifuged. The resultant supernatant was then added to 300 AL j nant breast cells and provide mechanistic insight into the etiology of glutathione-agarose beads, mixed on a rotating wheel at 4 C for 2 hours, of human breast cancer. and centrifuged. Bound GST-fused proteins in the pellet were washed five times with lysis buffer and resuspended in 300 AL of binding buffer [50 mmol/L NaCl, 20 mmol/L Tris-HCl (pH 8), 0.05% NP40, 0.25% bovine serum albumin, 1 mmol/L PMSF, 1 mmol/L DTT]. Bound protein was Materials and Methods quantitated with the Coomassie protein assay reagent (Pierce Co., Rockford, Cells and protein extracts. HepG2 human hepatoma cells, human IL), and 0.5 Ag of each GST-protein bound to glutathione-agarose beads was cervix epithelioid carcinoma cells (HeLa), and human normal and breast incubated with 7 ALofin vitro translated 35S-labeled protein in 150 ALof cancer cells (HBL-100, MCF-7, MDA-MB 231, MDA-MB 468, and MDA-MB binding buffer at 4jC for 2 hours. Reactions were terminated by 453; American Type Culture Collection, Manassas, VA) were maintained in centrifugation; the precipitate was washed thrice with protein binding DMEM (Life Technologies, Inc., Carlsbad, CA) supplemented with 10% fetal buffer and subjected to a 10% acrylamide SDS-PAGE; and proteins were bovine serum. Specimens of tissue from patients with breast cancer were visualized by autoradiography. collected intraoperatively and frozen in liquid nitrogen. Tumor tissue with Oligonucleotides and gel shift analysis. Wild-type and mutant C2 increased IR expression was selected and analyzed for each patient together (300 bp) sequence of the human IR promoter gene was generated by PCR with the healthy surrounding tissue. Nuclear and cytoplasmic extracts were amplification using the previously described recombinant plasmid pCAT-C2 prepared from cultured cells and breast tissue as described previously (11). (24) and its mutagenized clones as template (see below) and the following For each extract, an equal number of nuclei were homogenized, and the primers: C2 forward, 5¶-TCGAGTCACCAAAATAAACAT-3¶ (for wild-type final protein concentration in the extracts was determined by the modified and mutated oligonucleotides); C2 reverse, 5¶-TGCAGGGGAGGGAGGT- Bradford method (Bio-Rad, Hercules, CA). GCCGC-3¶ (for wild-type oligonucleotides); pCAT-C2 reverse, 5¶-ATTG- Reverse transcription-PCR, immunoprecipitation, and Western blot GGGATATATCAACGGTGGTATATCC-3¶ ( for mutated oligonucleotides). analysis. Total cellular RNA was extracted from cells and tissues using the 32P-labeled C2 was used in gel shift assays as previously described (11). RNAqueous-4PCR kit and subjected to DNase treatment (Ambion, Austin, Double-stranded 26-mer oligonucleotides containing wild-type or mutated TX). cDNA was synthesized from total RNA with the RETROscript first binding sites for AP-2a (Santa Cruz Biotechnology) were used in strand synthesis kit (Ambion) and used for PCR amplification. PCR competition studies and decoy experiments. products were electrophoretically resolved on 2% agarose gel, transferred to Chromatin immunoprecipitation. Chromatin immunoprecipitation GeneScreen-plus nylon membranes (DuPont, Fayetteville, NC), and assay was done as described previously (13), using HepG2 cells (1 Â 107) hybridized with 32P-labeled PCR-generated probes for the IR, AP-2a, transfected with the pC-hu/AP-2a expression vector. Formaldehyde-fixed C/EBPh, and tubulin. For quantitation of specific mRNA, hybridized DNA-protein complex was immunoprecipitated with anti-HMGA1, anti- membranes were exposed to X-ray film as needed to provide adequate Sp-1, or anti-AP-2a antibody. Primers for the human IR promoter sequence intensity of bands, and the band intensities of the autoradiograms were (ref. 24; forward, 5¶-AACCACCTCGAGTCACCAAAA-3¶ and reverse, 5¶-AGAG- quantified by densitometry. The values obtained were then normalized to GGAGGGAAAGCTTGCAG-3¶) were used for PCR amplification of immuno- those of the tubulin and C/EBPb genes. Western blot analyses of IR, AP-2, precipitated DNA (30 cycles), using PCR ready-to-go beads (Amersham and C/EBPh were done on total cellular lysates or nuclear extracts from Biosciences, Piscataway, NJ). PCR products were electrophoretically cells and tissues as previously described (11–13). For immunoprecipitation resolved on 1.5% agarose gel and visualized by ethidium bromide staining. studies with HMGA1 or Sp1 antibody, aliquots of HeLa or HepG2 nuclear Plasmids and mutagenesis. Eukaryotic expression plasmids used in extract were incubated for 12 hours with rotation at 4jC with 10 ALof this study were as follows: pcDNA1-HMGA1 (12), pEVR2/Sp1 (12), pC-hu/ antibody-coupled protein A beads. Beads were recovered by gentle AP-2a (a kind gift from Ko Fujimori), full-length and various truncated centrifugation and washed thrice with 500 AL of NETN wash buffer [0.1% forms of AP-2a (25). Site-directed mutagenesis of DNA binding sites for NP40, 150 mmol/L NaCl, 1 mmol/L EDTA, 50 mmol/L Tris-HCl (pH 8)] for HMGA1 and/or Sp1 in pCAT-C2 was done by the overlap extension method 5 minutes. Protein was removed from the beads by boiling in sample buffer (26), using wild-type C2 sequence as initial template in PCRs and the for 5 minutes and analyzed by SDS-PAGE and immunoblotting. Antibodies following primers (Life Technologies): C2-HMGA1 forward, 5¶-GCCCACT- used for these studies were as follows: anti-HMGA1 (11), anti-Sp1 (PEP 2; ATGAACCCAATAGCAACCTGGTAGAGAAAGG-3¶ and C2-HMGA1 reverse,

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5¶-CCTTTCTCTACCAGGTTGCTATTGGGTTCATAGTGGGC-3¶; C2-Sp1 for- mRNA levels were higher in certain human breast cancer cell lines ward, 5¶-CCCGGCACAGGGAGGCTTGGAGACGTGCGGGGCG-3¶ and C2-Sp1 (MCF-7, MDA-MB 231, and MDA-MB 468) compared with cells ¶ ¶ reverse, 5 -CGCCCCGCACGTCTCCAAGCCTCCCTGTGCCGGG-3 (first round); derived from normal human breast (HBL-100; Fig. 1). In addition, ¶ C2-Sp1 forward, 5 -GACGTGCGGGGCTTGGCGGGACCGAGCTGCACCTCC- IR mRNA levels in breast cancer specimens from six unrelated CTCC-3¶ and C2-Sp1 reverse, 5¶-GGAGGGAGGTGCAGCTCGGTCCCGCCAA- patients were significantly higher than those observed in the GCCCCGCACGTC-3¶ (second round). Mutagenized bases are in lowercase letters. Mutations were confirmed by sequence analysis. corresponding surrounding normal tissues (Fig. 1). IR mRNA Transcription studies. Recombinant plasmid pCAT-C2 containing wild- abundance in cultured breast cancer cells and tissues paralleled IR type or mutant DNA binding sites for HMGA1 and/or Sp1 and effector protein expression levels (Fig. 1). As measured by reverse vectors for AP-2, HMGA1, or Sp1 were transiently transfected into HepG2 transcription-PCR, the abundance of IR mRNA in breast cancer or MCF-7 cells by the calcium phosphate precipitation method, and CAT cell lines and tumoral breast tissues correlated closely with AP-2 activity was assayed 48 hours later as previously described (24). As an mRNA. The increase in AP-2 mRNA levels in both cells and tissues internal control of transfection efficiency, h-galactosidase activity was paralleled the increase in AP-2 protein expression levels. These measured in addition to protein expression levels (11). For decoy findings indicate that an up-regulation of AP-2 may occur in experiments, MCF-7 cells were cotransfected with pCAT-C2 reporter vector human breast cancer, and this overexpression correlates closely plus 20 Ag of double-stranded oligonucleotides containing wild-type or with IR mRNA and protein expression levels. mutated AP-2 binding site sequences and chloramphenicol acetyltransfer- ase (CAT) activity was measured as described above. Small interfering RNA AP-2 interacts with HMGA1 in the absence of DNA. We (siRNA) targeted to HMGA1 (ACCACCACAACTCCAGGAA), Sp1 (AATGA- previously showed that the architectural transcription factor GAACAGCAACAACTCC), and AP-2a (siGENOME SMART pool reagent), HMGA1 plays significant molecular roles in the context of the plus nonspecific siRNA controls with a similar GC content (NS-IX and NS-X) human IR gene, whose transcriptional activation is dependent on were obtained from Dharmacon (Lafayette, CO); 100-200 pmol siRNA the formation of a multiprotein-DNA complex, in which protein- duplex was transfected into cells at 50% to 60% confluency using protein and DNA-protein interactions are needed for transcrip- LipofectAMINE 2000 reagent (Invitrogen, Carlsbad, CA), and cells were tional synergism between the various transcription factors in vivo analyzed 48 to 96 hours later. HeLa nuclear extracts were employed in (11–13). As stated above, there are no AP-2 DNA-binding sites on in vitro in vitro transcription studies using the HeLa cell extract the promoter region of the IR gene. Therefore, in an attempt to transcription kit (Promega) plus the linearized pCAT-IR plasmid (24) as identify the biochemical mechanisms underlying the stimulatory the DNA template. Hybridization of RNA transcripts with a 32P-labeled CAT primer and reverse transcription were carried out as reported previously role of AP-2 in IR gene expression, we did experiments designed to (27). For competition experiments, double-stranded oligonucleotides investigate whether HMGA1 was necessary for recruitment and containing wild-type or mutated HMGA1 and/or Sp1 or AP-2 binding sites binding of AP-2 to this nucleoprotein complex controlling IR gene were added to untreated HeLa extracts, and the extracts were incubated for transcription. Direct physical association between HMGA1 and 15 minutes at room temperature before exposure to the IR-CAT template. multiple transcription factors has been reported (29, 30). To (Note: Sequences for non-indicated oligonucleotide primers are available analyze the ability of HMGA1 and AP-2 to interact with each other upon request.) in vitro, in the absence of DNA, we first did a GST pull-down assay, in which in vitro translated 35S-labeled AP-2 was analyzed for its ability to be specifically retained by a GST-HMGI affinity Results resin. As shown in Fig. 2A, AP-2 was retained by GST-HMGI but Expression of IR and AP-2 in breast cancer cells and tissues. not GST alone, suggesting that AP-2 interacts physically with Increased IR protein content in cultured human breast cancer cells HMGA1 in vitro. A bona fide interaction between HMGI and and whole breast tumor tissues has been reported earlier (7, 28). In AP-2 was observed in the presence of high concentrations of agreement with these previous observations, here, we show that IR ethidium bromide (not shown), which has been shown to disrupt

Figure 1. Comparison of IR mRNA and protein content with AP-2a mRNA and protein expression. A, mRNA abundance of each gene was quantified by reverse transcription-PCR in cultured cell lines (left) and breast tissues (right). B, total cell and nuclear extracts from cultured cells (left) and tissues (right) were resolved by SDS-PAGE, and IR and AP-2 protein expression levels were measured by immunoprecipitation and Western blot (IP/WB) with the anti-IRh antibody and the anti-AP-2a antibody. C/EBPh and tubulin, controls. T, tumoral; N, normalbreast tissues. Columns, quantitation of RT-PCR from cancer and normaltissues. Normalized mRNA is expressed as % maximal value (100%). n,IR; , AP-2. P < 0.001 compared with cancer group mean (unpaired Student’s t test). www.aacrjournals.org 5087 Cancer Res 2006; 66: (10). May 15, 2006

Figure 2. Physicalassociation between HMGA1 and AP-2a. A, SDS-PAGE of 35S-AP-2 bound to GST-HMGA1 resin. Lane 2, labeled protein was added directly onto the gelwithout binding to and elution from GST protein resin. B, immunoprecipitation (IP) of AP-2 and HMGA1 by using the anti-HMGA1 antibody followed by immunoblotting with the anti-AP-2 antibody (lanes 1-4), or the anti-HMGA1 antibody (lanes 5-8), after reprobing the same transfer. Lane 1, 10 ng of pure HMGA1; lanes 2 and 3, HeLa nuclear extract (NE; 500 Ag). Lanes 1 and 2, protein was directly applied to the gelwithout binding to and elutionfrom protein A beads. To prove specificity, pure Sp1 (lane 4, control) was used for immunoprecipitation by the anti-HMGA1 antibody. C, localization of the interacting domains within HMGA1 and AP-2. Wild-type 35S-labeled AP-2 (left) and its mutant derivatives (right) were incubated with resins containing normalized amounts of wild-type (1-107) and various mutant versions of GST-HMGI. Specifically, bound proteins were resolved by SDS-PAGE and visualized by autoradiography.

DNA-dependent protein-protein contact (31). Interaction between minimal region required for specific interaction with AP-2 HMGA1 and AP-2 was further investigated in coimmunoprecipi- corresponds to the middle basic repeat flanked by the spacer tation studies with cell nuclear extract and an antibody against region between the middle and the third basic repeats (amino acids HMGA1 immobilized on protein A beads. As shown in Fig. 2B, 54-74), revealing, in this respect, a behavior similar to that shown immunoprecipitation of HMGA1 from HeLa nuclear extracts by the interactions of HMGA1 with nuclear factor-nB (27), Sp1, and followed by Western blot analysis for AP-2 revealed a major C/EBPb (12). As a second step to characterize the AP-2 protein specific band, which migrated in a position corresponding to the domains involved in HMGA1 binding, similar experiments were size of AP-2. When the same transfer was reprobed with the anti- done with GST-HMGI wild-type and in vitro synthesized 35S-labeled HMGA1 antibody, a unique specific band, which migrated in a AP-2 mutants. As shown in Fig. 2C, full-length AP-2 and its mutant position corresponding to the size of HMGA1, was detected. Taken DN165 were both specifically retained by the agarose beads together, these data unequivocally indicate that HMGA1 and AP-2 coupled with GST-HMGI but not by GST alone. In addition, AP-2 physically interact either in vitro or in vivo in the context of the mutants DN227 and DC390 were not bound by GST-HMGI. These intact cell and suggest that physical and functional cooperation data show that binding of AP-2 to HMGI does not need the NH2- between HMGA1 and AP-2 on the IR promoter might occur terminal region upstream amino acid 165. Instead, the simulta- through direct contact as well. neous presence of both regions 165-227 and the COOH-terminal To elucidate which regions of HMGA1 and AP-2 were required domain downstream amino acid 390 of AP-2 are required for AP-2- for physical interactions, we first carried out GST pull-down assays HMGA1 interaction. Thus, these findings indicate that AP-2 using GST-linked deletion mutant versions of HMGI. As shown in interacts with HMGA1 through few multiple functional domains Fig. 2C, HMGI mutant 1-74, which lacks the COOH-terminal tail that can function as potent activators of transcription in the plus the third basic repeat, displayed an evident binding activity for presence of HMGA1. AP-2 similar to that of the wild-type (1-107) protein. No binding AP-2 interacts with Sp1 in the absence of DNA. A functional activity was detected with mutants 1-54 and 65-107 containing the interplay among AP-2 and Sp1 family factors has been shown in first and third basic repeats, respectively. Conversely, binding various pathways during activation of gene transcription in activity was detected within the HMGI mutant 54-74, containing eukaryotes (32, 33). On the other hand, functional cooperation the second basic repeat, whereas HMGI mutants lacking any basic between Sp1 and HMGA1 for the transcriptional activation of the repeat (clones 31-54 and 65-74) were unable to bind AP-2. Taken IR promoter has been reported previously by our group (12). To together, these data indicate that, in the context of HMGA1, the gain support for the assumption that AP-2 and Sp1 interact directly

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2006 American Association for Cancer Research. AP-2 and IR Overexpression in Breast Cancer at the IR promoter, we first tried to determine whether the two factors bind to each other in solution. To this end, a pull-down assay using GST-fused Sp1 and in vitro synthesized 35S-labeled AP-2 was done. As shown in Fig. 3A, AP-2 was efficiently retained by wild-type GST-Sp1 but not GST alone. To verify that the interaction observed in vitro between AP-2 and Sp1 resembles the interaction in vivo, we did coimmunoprecipitation studies using nuclear extracts from HepG2 cells cotransfected with expression vectors for AP-2 and Sp1, in the presence of an antibody against Sp1 immobilized on protein A beads. As shown in Fig. 3B, immunoprecipitation of Sp1 from nuclear extracts of transfected HepG2 cells followed by Western blot analysis for AP-2 revealed a unique specific band, which migrated in a position corresponding to the size of AP-2. When the same transfer was reprobed with an anti-Sp1 antibody, a major band that migrated in a position corresponding to the size of Sp1 was observed. Thus, these findings Figure 4. Physicalassociation between HMGA1, Sp1 and AP-2 a in vivo. in vivo Immunoprecipitation (IP) of HMGA1, Sp1, and AP-2 was carried out in nuclear indicate that these two factors physically interact . Further extracts (500 Ag) from untransfected (U) and transfected (T) HepG2 cells, using evidence of the interaction between the transcription factors the anti-HMGA1 antibody, followed by immunoblotting with the anti-HMGA1 investigated in this study was obtained by performing coimmuno- antibody (lanes 1 and 2), the anti-Sp1 antibody (lanes 3 and 4), or the anti-AP-2 antibody (lanes 5 and 6). The specificity of the immunoprecipitation with the precipitation experiments with nuclear extracts from transfected anti-HMGA1 antibody was tested with pure Sp1 alone (lane 7, control). HepG2 cells and the antibody against HMGA1 immobilized on protein A beads. As expected, immunoprecipitation of HMGA1 followed by immunoblot analyses with specific antibodies allowed the identification of three proteins, including, in addition to HMGA1, Sp1 and AP-2 (Fig. 4). Therefore, we conclude that a physical complex consisting of HMGA1, AP-2, and Sp1 exists in vivo in the context of the intact cell. AP-2 interacts with HMGA1 and Sp1 in the presence of DNA sequences for the IR promoter. Physical interaction among AP-2, HMGA1, and Sp1 has been characterized further by gel shift assay, using the previously described radiolabeled fragment C2 of the human IR promoter gene (24). Whereas binding sites for Sp1, flanked by AT-rich HMGA1 binding sites, have been identified within this DNA region, no putative binding sites for AP-2 have been detected within this element of the IR promoter. As shown in Fig. 5A, the binding of either pure recombinant HMGA1 (11) or pure Sp1 (a kind gift from Robert Tjian) to this probe produced a protein-DNA complex that was recognized and supershifted to a slower-migrating form by the anti-HMGA1 or anti-Sp1 antibody, respectively. Nuclear extracts from HepG2 cells (which do not express AP-2) transfected with AP-2 expression plasmid were used as a source of AP-2 nuclear protein. The incubation of nuclear extracts from transfected HepG2 cells with C2 probe yielded a higher protein-DNA complex that was in part recognized and supershifted by the anti-AP-2 antibody. The two additional complexes, other than that including AP-2, in HepG2 nuclear extracts, included Sp1 and HMGA1. The existence of AP-2 in a cocomplex with HMGA1 and Sp1 was confirmed in competition studies using nuclear extracts from HeLa cells, a cell line naturally expressing either HMGA1, Sp1, or AP-2 nuclear protein. In HeLa nuclear extracts, the formation of a large complex involving the simultaneous presence of all three proteins on the same probe C2 was prevented by using relatively small amounts of nuclear extracts Figure 3. Physicalassociation between AP-2 a and Sp1. A, SDS-PAGE of in the presence of increasing concentrations of competitor DNA for 35S-AP-2 bound to GST-Sp1 resin. Lane 2, labeled protein was added directly onto the gelwithout binding to and elutionfrom GST protein resin. AP-2 (Fig. 5A). Thus, these results show that AP-2 did not bind C2 B, immunoprecipitation (IP) of AP-2 and Sp1 by using the anti-Sp1 antibody directly but rather forms a multiprotein-DNA complex with Sp1 followed by immunoblotting with the anti-AP-2 antibody (lanes 1-4), or the anti-Sp1 antibody (lanes 5-8), after reprobing the same transfer. Lane 1, 5ngof and HMGA1 independently of AP-2 binding to DNA. These data pure Sp1; lanes 2 and 3, nuclear extract (NE; 500 Ag) from HepG2 cells were substantiated by chromatin immunoprecipitation assay, cotransfected with expression plasmids encoding AP-2 or Sp1. Lanes 1 and 2, showing that HMGA1, Sp1, and AP-2 indeed bind to the protein was directly applied to the gel without binding to and elution from protein IR in vivo a A beads. To prove specificity, pure HMGA1 (lane 4, control) was used for endogenous locus in AP-2 -overexpressing HepG2 cells immunoprecipitation by the anti-Sp1 antibody. (Fig. 5B). www.aacrjournals.org 5089 Cancer Res 2006; 66: (10). May 15, 2006

Figure 5. HMGA1, Sp1, and AP-2 are associated with the IR promoter in vitro and in vivo. A, gel shift assay of radiolabeled fragment C2 (0.2 ng) with 2.5 ng of either HMGA1 (lane 2) or pure Sp1 (lane 4), in the presence of 2.0 Ag of bovine serum albumin or 0.5 Ag of nuclear extracts (NE) from untransfected (lane 6) and transfected (lane 7) HepG2 cells and 0.2 Ag of poly(deoxyinosinic-deoxycytidylic acid) as the competitor DNA. In supershift assays the protein was preincubated with 1 Ag of polyclonal antibody to HMGA1 (lane 3), Sp1 (lane 5), or AP-2 (lane 8) before addition of the probe. Protein binding activity of probe C2 was analyzed in competition assays using nuclear extract (NE) from HeLa cells, in the presence of increasing amounts of unlabeled oligonucleotides bearing either a wild-type (AP-2wt, 0-200 molar excess, lanes 10-13) or mutant (AP-2m, 200 molar excess, lane 14) binding sequence for AP-2. Lanes 1 and 9, probe alone. B, chromatin immunoprecipitation of the IR promoter gene in HepG2 cells untransfected (U) or transfected (T) with AP-2a expression vector. Chromatin immunoprecipitation was done using antibodies (Ab) against either HMGA1, Sp1, or AP-2a.

Induction of the IR promoter by AP-2A requires HMGA1 in these cells led to a significant increment in CAT activity that and Sp1. In the light of the above results indicating that AP-2 exceeded that seen with either factor alone and required intact physically interacts with HMGA1 and Sp1, it was important to see binding sites for HMGA1 and Sp1, as shown by diminished activity whether these interactions had a functional role in the transcrip- of constructs mutated at either or both of these sites. Induction of tional activity of the IR gene promoter. The effect of AP-2a on IR CAT activity was at least in part dependent on endogenous AP-2, as transcription was first evaluated by transiently transfecting the revealed by AP-2 decoy (36) and siRNA strategies. Cotransfection of AP-2-null cell line HepG2 (naturally expressing both HMGA1 and a cis element decoy against the AP-2 binding site resulted in a 50% Sp1) with the C2 IR promoter element (pCAT-C2) in the presence reduction in CAT activity, whereas transfection of the mutated AP-2 of an expression vector encoding the wild-type AP-2a. As shown decoy element, which fails to bind the AP-2 protein in vitro, was in Fig. 6A, overexpression of AP-2 in HepG2 cells induced a ineffective. siRNA-mediated knockdown of endogenous AP-2a in concentration-dependent stimulation of IR gene transcription. MCF-7 cells showed similar results (Fig. 6C). In concert with these Because AP-2 did not bind the IR promoter element directly, findings, endogenous IR expression was reduced in MCF-7 cells this result supports a model in which AP-2 activates IR gene subjected to siRNA-mediated knockdown of AP-2a (Fig. 6D). These transcription independently of AP-2 binding to DNA. To examine results were supported further by in vitro transcription studies the possibility that transactivation of the IR promoter by AP-2 using HeLa nuclear extracts in the presence of synthetic oligo- could involve HMGA1 and Sp1, we generated CAT constructs with nucleotides with either the HMGA1, Sp1, or AP-2 binding sequence the C2 regulatory element mutated at either or both of the HMGA1 (Fig. 7). Together, these data indicate that AP-2a plays a positive and Sp1 binding sites. Mutation of the HMGA1 or Sp1 binding site role in IR gene expression and suggest that AP-2a may regulate IR in the C2 promoter element (pCAT-C2/HMGA1m or pCAT-C2/ promoter activity through direct association with HMGA1 and Sp1 Sp1m, respectively) reduced CAT activity in AP-2-transfected rather than binding to DNA. HepG2 cells to 15% to 20% of that with the native (pCAT-C2) promoter. Mutation of both binding sites together (pCAT-C2/ HMGA1m/Sp1m) almost completely abolished CAT activity. Discussion Likewise, IR-CAT activity was impaired in HepG2 cells transfected Breast cancer is an important public health problem because it with AP-2a mutant effector plasimds (AP-2aDC390, AP-2aDN165, affects about one in 10 women at some time during their lives and and AP-2aDN227), providing further evidence of the existence of represents a leading cause of cancer deaths among women. A wide important functional interactions among HMGA1, Sp1, and AP-2 variety of studies have shown that the IR is frequently overex- in the context of the IR gene (Fig. 6A). Consistent with these pressed by most human breast cancers, suggesting a role for this observations, endogenous IR protein and mRNA (data not shown) tyrosine kinase receptor protein in breast cancer cell biology. levels were very low in AP-2a-overexpressing HepG2 cells subjected Furthermore, ligand-dependent malignant transformation and to siRNA-mediated knockdown of HMGA1 and/or Sp1 (Fig. 6B). increased cell growth occur in cultured breast cells overexpressing Finally, we showed that the interplay among AP-2, HMGA1, and IRs. The IR can exert its oncogenic potential in malignant breast Sp1 had functional sequelae also in MCF-7 cells, a cell line ideally cells via abnormal stimulation of multiple cellular signaling suited for studying the effects of these proteins on transcription cascades, enhancing growth factor–dependent proliferation and/ because it does not express appreciable levels of either HMGA1 or or by directly affecting cell metabolism. However, little is known Sp1 (34, 35), whereas AP-2 is constitutively expressed (21). As about the molecular mechanisms that account for the increased IR shown in Fig. 6C, simultaneous overexpression of HMGA1 and Sp1 expression in human breast cancer.

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In target cells, the IR has been shown to be under the regulation lines and breast cancer tissues. Activation of gene expression by of hormones, metabolites, and differentiation (24). The transcrip- AP-2 has been reported for many different genes, in which AP-2 tion of the IR gene is also dependent upon transcription factor DNA binding sites have been identified in the promoter region of Sp1 (12). Our data implicate AP-2a in the up-regulation of IR the gene (37). In our study, we provide compelling evidence that expression in breast cancer, and this up-regulation may be increased IR expression in breast cancer is dependent on the important for the development of malignant transformation. As assembly of a transcriptionally active multiprotein-DNA complex, shown by immunoblot and gene expression analyses, AP-2a was which includes, in addition to AP-2, the architectural factor significantly increased in nuclear extracts from cultured breast HMGA1 and the ubiquitously expressed transcription factor Sp1. cancer cells and human breast cancer specimens, and this increase The observation that AP-2 forms a protein-protein complex with correlated closely with IR overexpression in both breast cancer cell HMGA1 and Sp1 in the activation of IR promoter, in the absence of

Figure 6. Functionalsignificance of AP-2 a for the IR gene transcription. A, HepG2 cells were transfected with CAT reporter plasmids (2 Ag) containing wild-type (wt)or mutant (m) versions of the C2 IR promoter element, in the absence or presence of wild-type or various mutant derivatives of AP-2a effector plasmid (5 Ag). White columns, mock (no DNA); black columns, pCAT-basic (vector without an insert). B, wild-type AP-2a expression plasmid was transfected into HepG2 cells. After 6 hours of transfection, the cells were treated with anti-HMGA1 (100 pmol) and/or anti-Sp1 (200 pmol) siRNA or a nontargeting control siRNA, and endogenous IR protein expression was measured 48 to 96 hours later. C, MCF-7 cells were transfected as in (A), along with HMGA1 and/or Sp1 expression plasmids (5 Ag each), in the absence of AP-2 expression vector. For decoy experiments, cells were cotransfected with a double-stranded oligonucleotide containing the wild-type (AP-2wt) or mutant (AP-2m) binding sequence for AP-2a and CAT activity was measured 48 hours later. Specific knockdown of AP-2a was achieved by transfection of an anti-AP-2a siRNA (100 pmol), and CAT activity was measured as above. D, MCF-7 cells were transfected with anti-AP-2a siRNA or a nontargeting controlsiRNA, and the endogenous IR protein expression was measured as above. Columns, means for three separate experiments; bars, SE. CAT values are expressed as the factor of increase above the level of CAT activity obtained in transfections with wild-type reporter vector alone, which is assigned an arbitrary value of 1. IR protein expression is shown as percentage of the expression in the presence of the siRNA control(100%). Western blots of HMGA1, Sp1, and AP-2 are shown in the autoradiograms.

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represents a fundamental prerequisite for AP-2a to properly stimulate IR gene transcription in the absence of specific AP-2 consensus binding sites. This result closely resembles a previous one for transcriptional activation of the CYP11A1 gene, in which a functional interplay between AP-2 and Sp1 has been shown in the context of the CYP11A1 gene promoter, in the absence of an AP-2 binding site (33). Stabilization of transcription factor-DNA interactions by HMGA1 plays a critical role in gene regulation. In particular gene contexts, HMGA1 acts to stabilize transcription factor-DNA and protein- Figure 7. Functionalsignificance of HMGA1, Sp1, and AP-2 for IR gene protein interactions through a combination of its own binding to transcription in HeLa nuclear extract. In vitro IR gene transcription was a proximal minor groove AT-rich sequence and through direct measured in HeLa nuclear extract, in the absence or presence of oligonucleotides (10 ng each) bearing either a wild-type (wt) or mutant (m) interaction with an adjacent transcription factor through one of its binding sequence for HMGA1, Sp1, or AP-2. additional AT-hook motifs or through direct effects on the con- formation of multisubunit proteins (14–17, 42). Our data suggest IR a AP-2 DNA-binding sites, is consistent with previous studies that induction of gene transcription by AP-2 in breast cancer can showing that AP-2 is capable of regulating gene transcription occur independently of AP-2 binding to DNA and support a model in indirectly, through the formation of heteromeric protein-protein which HMGA1 may facilitate and stabilize interactions between interactions between AP-2 and c-Myc (38) and AP-2 and E1A (39), AP-2 and Sp1 in the preinitiation complex at the IR promoter. To in the absence of AP-2 protein-DNA interaction. In addition, our our knowledge, this is the first report describing a quantitative observation agrees closely with the results reported in previous abnormality in a trans-acting factor, which affects the levels of IR works, indicating that transcriptional activation by AP-2 may expression of the gene in human breast cancer. We believe that a require combinatorial mechanisms, as the DNA sequences impli- AP-2 overexpression leading to IR overexpression might be a a cated in transactivation by AP-2 do not always resemble an AP-2 critical event in breast carcinogenesis, and that AP-2 is a potential binding site, and the proline-rich activation domain of AP-2 is a target for therapeutic intervention in human breast cancer. poor transactivator when positioned in a distal position (33, 38, 40). High levels of AP-2 protein expression in human breast cancer Acknowledgments have been reported in previous reports, in which expression of AP-2 correlated with the regulation of multiple growth factor signaling Received 10/11/2005; revised 1/22/2006; accepted 3/13/2006. a Grant support: Ministero dell’Universita`e della Ricerca, Italy protocol 2004062059- pathways (22, 41). Herein, we show for the first time that AP-2 002 and Telethon, Italy grant GGP04245 (A. Brunetti). induces transcriptional activation of the IR gene, and this The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance activation occur independently of AP-2 binding to the IR promoter. with 18 U.S.C. Section 1734 solely to indicate this fact. As shown in transient transfection studies using the AP-2-null cell We thank Ko Fujimori (Osaka Bioscience Institute), Tom Maniatis (Harvard line HepG2, transactivation of the IR promoter by AP-2 required University, Cambridge, MA), Hans Rotheneder (University of Vienna), Guntram Suske (Philipps University Marburg, Marburg, Germany), Dimitris Thanos (Columbia intact DNA binding sites for HMGA1 and Sp1 and needed direct University), Robert Tjian (University of California, Berkeley), and Erhard Wintersberger physical association of AP-2 with HMGA1 and Sp1, which (University of Vienna) for providing reagents and Anna Malta for secretarial help.

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