Taf4b and Jun/Activating Protein-1 Collaborate to Regulate the Expression of Integrin Α6 and Cancer Cell Migration Properties

Published OnlineFirst March 30, 2010; DOI: 10.1158/1541-7786.MCR-09-0159

Molecular Signaling and Regulation Cancer Research TAF4b and Jun/Activating Protein-1 Collaborate to Regulate the Expression of Integrin α6 and Cancer Cell Migration Properties

Margarita Kalogeropoulou1, Angeliki Voulgari1, Vassiliki Kostourou2, Raphael Sandaltzopoulos3, Rivka Dikstein4, Irwin Davidson5, Laszlo Tora4, and Alexander Pintzas1

Abstract The TAF4b subunit of the transcription factor IID, which has a central role in transcription by polymerase II, is involved in promoter recognition by selective recruitment of activators. The activating protein-1 (AP-1) family members participate in oncogenic transformation via gene regulation. Utilizing immunoprecipitation of endogenous protein complexes, we documented specific interactions between Jun family members and TATA box binding protein–associated factors (TAF) in colon HT29 adenocarcinoma cells. Particularly, TAF4b and c-Jun were found to colocalize and interact in the nucleus of advanced carcinoma cells and in cells with epithelial-to- mesenchymal transition (EMT) characteristics. TAF4b was found to specifically regulate the AP-1 target gene involved in EMT integrin α6, thus altering related cellular properties such as migration potential. Using a chromatin immunoprecipitation approach in colon adenocarcinoma cell lines, we further identified a synergistic role for TAF4b and c-Jun and other AP-1 family members on the promoter of integrin α6, underlining the existence of a specific mechanism related to gene expression control. We show evidence for the first time of an interde- pendence of TAF4b and AP-1 family members in cell type–specific promoter recognition and initiation of transcription in the context of cancer progression and EMT. Mol Cancer Res; 8(4); 554–68. ©2010 AACR.

Introduction apoptosis (5, 6), cancer, and epithelial-to-mesenchymal transition (EMT; ref. 7). TAF4b was first identified as a The binding of the transcription factor IID (TFIID) tissue-specific TFIID subunit, present only in a limited complex, composed of the TATA box-binding protein number of complexes, and was later shown to be necessary (TBP) and 14 TBP-associated factors (TAF), to promoter for ovarian follicle development, proliferation, and function DNA is responsive to cellular signals and constitutes the (8). TAF4b shares high homology with the COOH-termi- first step in transcription. A number of different TFIID nal part of TAF4 in contrast to its coactivator NH2-terminal forms with functionally distinct properties exist, among domain (9). TAF4b contains a nuclear export signal allowing which the TAF10-free TFIID, the TAF4b-containing it to shuttle between the nucleus and the cytoplasm (10), TFIID, the TAF6δ-containing TFIID, the TBP-free although it displays DNA-binding capacity when incorpo- TFIID, and the seven TAF complex have been described rated into the TFIID (11). Even though there is no evidence (1, 2). Notably, several studies suggest that TAFs are im- for a direct sequence-specific contact between DNA and portant in specific events like cell cycle regulation (3, 4), TAF4b, the involvement of TAF4b in direct promoter- selective recognition and subsequent recruitment of activators in a cell type–specific manner has been suggested Authors' Affiliations: 1Laboratory of Signal Mediated Gene Expression, Institute of Biological Research and Biotechnology, National Hellenic (12). Indeed, transcriptional induction of the activating Research Foundation, Athens, Greece; 2Biomedical Sciences Research protein-1 (AP-1) family member c-Jun by TAF4b in gran- Center “Alexander Fleming”, Vari, Greece; 3Laboratory of Gene ulosa cells has recently been proposed (13). As a member Expression, Molecular Diagnosis, and Modern Therapeutics, Department of the AP-1 transcription factor, c-Jun participates in the of Molecular Biology and Genetics, Democritus University of Thrace, Dragana, Alexandroupolis, Greece; 4Department of Biological Chemistry, control of cellular responses, mainly by converting extra- Weizmann Institute of Science, Rehovot, Israel; and 5Department of cellular signals into specific gene expression profiles via the Functional Genomics, Institut de Génétique et de Biologie Moléculaire et general transcription machinery. Altering the transcrip- Cellulaire, CNRS UMR 7104, INSERM U 964, Université de Strasbourg, Illkirch Cedex, France tion of target genes, c-Jun has been shown to interact with Note: Supplementary data for this article are available at Molecular the coactivator CBP (14) and with TAF7 in HEK293 and Cancer Research Online (http://mcr.aacrjournals.org/). COS cells (15). Corresponding Author: Alexander Pintzas, National Hellenic Research c-Jun follows a two-stage activation pattern including a Foundation, 48 Vassileos Constantinou Avenue, 11635 Athens, Greece. step of phosphorylation by mitogen-activated protein ki- Phone: 30-21072-73753; Fax: 30-21072-73755. E-mail: [email protected] nases (ERK, JNK, p38) and a subsequent selective forma- doi: 10.1158/1541-7786.MCR-09-0159 tion of dimers whose nature defines the activation of a ©2010 American Association for Cancer Research. specific subset of AP-1 binding site containing target genes

554 Mol Cancer Res; 8(4) April 2010

TAF4b and c-Jun Regulate Cell Migration Properties

(16, 17). Notably, AP-1 activity is frequently elevated in SDS-PAGE and transferred to a nitrocellulose membrane transformed cell lines due to an oncogene-specific upregu- (Pall Corporation). The antibodies used for immunoblot- lation of the AP-1 family members c-Jun, JunB, Fra-1, and ting are described in Supplementary Data. Signals were vi- Fra-2 (18, 19). Different types of tumors have been related sualized using enhanced chemiluminescence (Amersham to RAS-protein activation, which in turn, regulate the ac- Biosciences) after exposure to Kodak Super RX film. All tivity of AP-1 (20). For instance, c-Jun, which is frequently experiments were repeated at least three times. Representa- implicated in the acquisition of invasive properties in ag- tive images are shown. gressive forms of cancer (21), is required for in vitro cellular transformation by oncogenic RAS partially via a phosphor- RNA Extraction and Reverse Transcription-PCR ylation mechanism (22, 23). RNA was prepared from sampled cells by the TRIzol re- Colorectal carcinogenesis occurs through the accumula- agent (Invitrogen). Reverse transcription was carried out tion of gene alterations in tumor suppressor genes and on- using the SuperScript Reverse Transcriptase (Invitrogen) cogenes including RAS (24), leading to invasion/metastasis and oligo(15)-(dT), following the instructions of the manu- (25). EMT, occurring during the last steps of cancer pro- facturer. Primers are described in the Supplementary Data. gression prior to metastasis, is controlled by a number of Values were measured using the Image-Quant software regulators resulting in a loss of cell-cell adhesion, mediated (Amersham Biosciences). All experiments were repeated at by repression of E-cadherin, whereas vimentin and other least three times. Representative images are shown. mesenchymal proteins like matrix metalloproteinases and fibronectin are upregulated (26). Importantly, activation Real-time PCR and maintenance of EMT can be achieved by the signaling Real-time quantification was carried out using a Bio-Rad cascade of an oncogenic form of Harvey RAS (Ha-RAS; iCycler and the iQ5 Multicolor Real-time PCR detection ref. 27). Even though the phenomenon of EMT reflects system (Bio-Rad). Cycling conditions included a denatur- a transient state in vivo, by constitutively expressing the ing step of 3 min at 95°C followed by 40 cycles at 95°C for mutated Ha-RASV12 in the intermediate colon adenoma 40 s and annealing/elongation at 60°C for 40 s. All genes Caco-2 cell line, we have created a cell line (Caco-H) were tested in triplicate. Values were normalized to glycer- which adopts and maintains an EMT state (28). aldehyde-3-phosphate dehydrogenase (GAPDH). Results In this study, focusing on the investigation of Jun family were analyzed on the iCycler software. members and their interplay with TAFs in colon cancer and metastasis, we have identified an interaction between c-Jun and TAF4b and have evaluated its effect in the reg- Immunoprecipitation μ ulation of integrin α6, an EMT-related AP-1 target gene. Nuclear protein extracts (100 g) were incubated over- μ The implication of other AP-1 family members in this night at 4°C under rotation with 5 g of c-Jun, JunB, mechanism suggests a dynamic switch between these pro- JunD, and TAF4b (9) antibodies in a total volume of μ teins and their interacting partners in the control of 500 L of 100 mmol/L NaCl immunoprecipitation buffer, μ transcription. adding 25 L of dry Protein A-Sepharose matrix CL-4B (Amersham Biosciences) over a period of 2 h, followed Materials and Methods by three washing steps with 500 mmol/L of KCl immunoprecipitation buffer and 100 mmol/L of KCl immunopre- Propagation and Treatment of Cell Lines cipitation buffer. To detect specific interactions with TAFs, Caco-2, HT29, and HCT116 cells were obtained Western blotting analysis of the immunoprecipitated com- from American Type Culture Collection and cultured plexes was done by immunoblotting with TAF antibodies. in DMEM supplemented with 10% fetal bovine serum, antibiotics, and nonessential amino acids (all from Invi- Chromatin Immunoprecipitations (ChIP) and trogen, Corp.). Caco-2 cells constitutively overexpressing Re-ChIPs HRASV12 (Caco-H) were cultured as mentioned above. The protocols used have been previously described (7). For reasons of consistency, the name Caco-H will be used Chromatin was incubated with 5 μg of anti-TAF4b (9) or throughout the text when referring to Caco-2 cell lines any other antibody, as indicated overnight at 4°C. For overexpressing Ha-RASV12 in the case of presenting re- Re-ChIP experiments, complexes were eluted after the sults of only one Caco-H cell line. Although in some ex- first round of immunoprecipitation by 30 min of incu- periments, two different Caco-H cell lines were presented bation at 37°C in 10 mmol/L of DTT. The eluted (referred to as Caco-H1 and Caco-H2) for the validation of chromatin was diluted 20 times in sonication buffer results. and again subjected to chromatin immunoprecipitation procedures with the indicated antibodies. De–cross- Protein Extraction, Western Blotting, and Antibodies linking of eluted chromatin was done by the addition Nuclear, cytoplasmic, and whole cell lysate extracts were of 200 mmol/L of NaCl plus RNase A overnight at prepared as described earlier (19, 29). Protein concentra- 65°C. The remaining proteins were digested with tions were determined by the Bradford method using a Proteinase K for 2 h at 42°C. DNA fragments were re- Bio-Rad protein assay kit. Extracts were subjected to covered by phenol/chloroform extraction.

www.aacrjournals.org Mol Cancer Res; 8(4) April 2010 555

Kalogeropoulou et al.

Confocal Laser Scanning Microscopy stranded oligos were blunt-ended (sequences of oligos in Cells on glass coverslips were fixed with 4% paraformal- Supplementary Data). dehyde for 10 min at room temperature. Cell membranes were permeabilized with 0.1% Triton X-100 in PBS for 15 Fluorescence-Activated Cell Sorting Analysis min. Blocking of cells was done with 5% fetal bovine se- Forty-eight hours after the transfection of cells with rum in PBS for 1 h at room temperature. The antibodies TAF4b siRNA or siRNA control, cells were harvested, and dilutions used are listed in the Supplementary Data. washed with PBS, and 1 μg of integrin α6 antibody (Santa Nuclei were stained with Hoechst 33258 (Sigma-Aldrich). Cruz Biotechnology) was added to 1 × 106 cells and kept The slides were viewed with a 40× objective, Leica TCS on ice for 30 min. After washing twice with PBS, cells were SPE confocal laser scanning microscope (Leica Leisertech- incubated for 30 min in secondary antibody (anti-mouse nik). The objective lens used was 63×. LAS AF software Alexa-488) and again washed twice with PBS before anal- was used for image acquisition. ysis using FACScan CANTO II (Becton Dickinson).

DNA Transfection Short Interfering RNA Transfection Proliferation Assay Plasmid DNA was transfected into cells by the calcium Forty-eight hours after TAF4b and TAF4 siRNA treat- phosphate method (30). For short interfering RNA (siRNA) ment, cells in 12-well plates were fixed with methanol, treatment, cells were transfected with human TAF4b siRNA, stained with 0.5% crystal violet, and washed with PBS. c-Jun siRNA, TAF4 siRNA, or with siControl using the Stained cells were extracted using 30% acetic acid. Absor- protocols of the manufacturer (Dharmacon). After trans- bance was measured at 595 nm. fection (48 and 72 h), cells were harvested and extraction of proteins and RNA was done. Statistical Analysis Data are represented throughout the text with ±SD error bars. Statistical significance was tested with unpaired Stu- Luciferase Reporter Assay dent's t test. To assess luciferase activity, the Promega Dual Luciferase Reporter Assay (Promega Corporation) was used according to the instructions of the manufacturer. Luminescence was Results measured using a Tecan Safire fluorescence plate reader (Tecan Group, Ltd.). The reporter plasmid used was In Human Colon Carcinoma Cells, c-Jun Specifically 5xcoll-TRE-tata-luciferase (31), whereas the expression Interacts with TAF4b plasmids included TAF4b (9), c-Jun (32), and TAF4 (33). To unveil any interactions between Jun family members and human TAFs possibly playing a role in carcinogenesis, – Cellular Migration Assays immunoprecipitations with anti c-Jun, JunB, and JunD The assays were done on transwell plates (Corning Co- antibodies were done using nuclear extracts from the hu- star, Co.). Twenty-four hours after transfection with man colon adenocarcinoma cell line HT29. Among the in- siRNA, treated cells (1 × 104) were trypsinized and migra- teractions identified (Table 1) by Western blot analysis, a tion ability was measured as described previously (7). Cells specific interaction between c-Jun and TAF4b was ob- were visualized and counted by bright-field microscopy served in the c-Jun immunoprecipitate, the specificity of which was tested by siRNA against c-Jun and subsequent with a Nikon Eclipse TE200 inverted fluorescence/phase – contrast microscope equipped with a Sony charge-coupled immunoprecipitation with anti c-Jun antibody (Fig. 1A). device camera with a 40× objective. Data were obtained Verifying this interaction, in the inverse situation, c-Jun from two independent experiments, each repeated twice.

Electrophoretic Mobility Shift Assay Table 1. TAFs tested in immunoprecipitations Nuclear extracts (8 μg) were incubated for 60 min at with anti–c-Jun, anti-JunB, and anti-JunD room temperature with a 32P-labeled double-stranded antibodies probe ([γ-32P]ATP; Perkin-Elmer) in the presence of 1.5 μg of poly(deoxyinosinic-deoxycytidylic acid; Sigma- TAFs Aldrich). The reaction was loaded onto native 6% poly- Interaction with TAF1, TAF3, TAF4, TAF4b TAF10 acrylamide gels containing 0.5× Tris-borate EDTA buffer. TAF5, TAF6α, TAF6δ, The gel was prerun for 30 min at 120 V, run at 250 V for TAF7, TAF9, TAF12 2 h, dried for 1.5 h, and exposed for 12 h to a storage c-Jun − + − phosphor screen (Amersham Biosciences). Scanning was JunB −−+ done with a Storm 860 scanner (Amersham Biosciences) JunD −−− and values were measured using ImageQuant software. – For competition, nonlabeled probe (100 molar fold excess NOTE: TAF4b was shown to interact with c-Jun and TAF10 of the labeled probe) was incubated with nuclear extracts with JunB. for 30 min before the addition of the labeled probe. Double-

556 Mol Cancer Res; 8(4) April 2010 Molecular Cancer Research

TAF4b and c-Jun Regulate Cell Migration Properties

mechanisms, we first analyzed the expression levels of the two proteins in Caco-2 and Caco-H cells. The levels of TAF4b in the total extracts showed small differences between the cell lines tested, whereas, as expected, c-Jun revealed an overexpression pattern in all cell lines as compared with Caco-2 (Fig. 2A). The levels of EMT mar- kers, E-cadherin and vimentin, were also evaluated and showed that these cell lines were at different stages of cancer progression. Overexpression of the Ha-RAS protein in Caco-H cell lines (Caco-H1 and Caco-H2) was confirmed. In parallel, a quantification of the mRNA levels of c-Jun showed that they were highly increased in both Caco-H cell lines as compared with Caco-2, whereas the mRNA levels of TAF4b, in agreement with its protein expression pattern, were slightly increased in Caco-H cells compared with the increase of c-Jun in the same cell lines (Fig. 2B). In an attempt to elucidate the specific role of TAF4b in cancer, we reduced the levels of TAF4b and its homologue TAF4 by using specific siRNA either against TAF4b or TAF4. Seventy-two hours after transfection, the reduction of the mRNA levels of TAF4b and TAF4 was measured by PCR analysis (Fig. 3A, left). Interestingly, the cells subjected to TAF4b siRNA treatment, but not those transfected with TAF4 siRNAs, acquired up to a 76% differentiation in appearance, forming prolonged cell protrusions (Fig. 3A, right and bottom). In both cases, cell proliferation was unaffected (Fig. 3B). Because this morphologic FIGURE 1. Specific interactions between Jun family members and TAFs change was predominant in cells with EMT, we investi- in colon adenocarcinoma cells. A, top, HT29 nuclear extracts treated with gated the role of TAF4b in related cell characteristics. Inter- siRNA control (siControl) or c-Jun–specific siRNA (siRNA c-Jun) were estingly, the migration ability of Caco-H cells in response used for immunoprecipitation with c-Jun antibody. The samples were to a chemoattractant gradient was increased by 80% after analyzed by Western blotting (WB) with anti–c-Jun and anti-TAF4b antibodies. Downregulation of c-Jun expression decreased the TAF4b siRNA treatment (Fig. 3C). Similarly, Caco-2 interaction complex between TAFb and c-Jun as compared with and colon cancer HCT116 cells with partial EMT charac- immunoprecipitation under siControl treatment. Western blotting with teristics in which c-Jun and TAF4n were also detected to anti-actin antibody was used for the control of equal protein loading. interact (Supplementary Data) showed increased migration Bottom, Western blotting analysis of immunoprecipitation with abilities of 50% and 30%, respectively, as compared with anti-TAF4b using HT29 nuclear extracts. B, Western blotting analysis of immunoprecipitation with anti–c-Jun using nuclear extracts from cells control cells or cells treated with siControl. Together, these expressing mutant Ha-RAS (Caco-H) as compared with their parental cell results indicate a negative role for TAF4b in cell migration line Caco-2. and metastasis-related phenomena. was detected in the TAF4b immunoprecipitate (Fig. 1A, TAF4b Regulates the Expression of Integrins bottom). We focused on this interaction as they have been Based on the proposed preferential regulation of AP-1 site– shown to influence each other during the induction of spe- containing genes by TAF4b and its implication in cell motil- cific transcription programs (13). To identify a potential ity, we focused our study on the search for a TAF4b-regulated role in cancer-related mechanisms, the same experiment gene playing a role in cell migration while bearing an AP-1 was done using nuclear extracts from parental Caco-2 site in its promoter. Changes in the mRNA levels of some and Caco-H cells with EMT characteristics. The same metastatic/EMTmarkers after knockdown of TAF4b by siR- interaction was confirmed and was slightly enhanced in NA were analyzed revealing increased levels of only some Caco-H cells (Fig. 1B). Together, these experiments not genes (e.g., MMP-2 and integrin α2; Supplementary Data). only show a specific interaction between TAF4b and Interestingly, a gene important for cell migration—also bear- c-Jun but also stress out the possible significance of this ing a putative AP-1 site in its promoter (34), i.e., integrin α6, interaction in EMT phenomena. after TAF4b siRNA treatment in Caco-2, Caco-H, and HCT116 cells—showed a decrease in its mRNA levels (Fig. 4A), surmising a regulatory function of TAF4b on its TAF4b Plays a Role in the Migration Ability of Cells transcriptional activation. Similarly, integrin α6 hemidesmo- To further explore the two components involved in the some partners integrin β4(Fig.4C)andintegrinβ1 (Fig. 4D) interaction and identify any possible role in cancer-related showed lower mRNA levels after TAF4b siRNA treatment.

www.aacrjournals.org Mol Cancer Res; 8(4) April 2010 557

Kalogeropoulou et al.

Notably, integrin α6 showed higher mRNA levels in the of its expression in the cell lines tested, similarly to TAF4b EMT-like Caco-H cells, which have also shown a greater (Fig. 5B). In addition, fluorescence-activated cell sorting extent of TAF4b/c-Jun colocalization (Fig. 2C) and analysis showed that, similar to TAF4b (Fig. 5C), c-Jun in the advanced colon cancer HCT116 cells (Fig. 4A, changed the localization of integrin α6 in the cell surface after left) as compared with Caco-2 cells. Interestingly, integrin treatment of cells with c-Jun–specific siRNA. This data α6 also showed higher protein levels in Caco-H and indicates that integrin α6, β1, and β4 are transcriptionally HCT116 cells as compared with Caco-2 cells, and the regulated by TAF4b, whereas a coinvolvement of AP-1 family highest overexpression rate as compared with integrin β4 member and TAF4b-interacting partner c-Jun in the tran- and β1 in the same cell lines (Supplementary Data). Im- scriptional regulation of integrin α6 has been proposed and munostaining of the integrin α6 protein confirmed the in- will be further studied. creased expression of integrin α6 in Caco-H cells, whereas visualizing its accumulation in cell protrusions (Fig. 4D, Complex Formation of c-Jun and TAF4b on the AP-1 right). At the same time, underlining the specificity of Site of the Integrin α6 Promoter In vivo and a Cell TAF4b on the regulation of integrin α6, TAF4 siRNA Type–Specific, c-Jun–Binding Dependence of TAF4b treatment in the same cells did not affect the mRNA levels To understand the importance of an AP-1 site for gene of integrin α6 or that of TAF4b (Supplementary Data). regulation by TAF4b, a luciferase reporter construct con- TAF4b-specific siRNA treatment, on the other hand, not trolled by the AP-1 site (5xcoll-TRE-tata-luciferase) of only reduced the levels of integrin α6 but also induced a the Collagenase gene promoter was activated after c-Jun change in its localization. As in cells under siControl treat- and TAF4b overexpression in Caco-2 cells. On the other hand, ment, the integrin α6 protein was localized mainly on the an overexpression of TAF4 did not produce the same effects, cell surface of Caco-H cells. In cells treated with siRNA suggesting a specific involvement of TAF4b in AP-1–binding against TAF4b, integrin α6 was found localized throughout complexes (Fig. 6A, left). Overexpression of the transfected the cell (Fig. 4E). The same was shown with fluorescence- genes was confirmed by Western blotting (Fig. 6A, right). activated cell sorting analysis comparing the cell surface ex- To investigate the c-Jun/TAF4b interaction and eluci- pression of integrin α6 in cells treated with siControl and date its possible effect on AP-1 site–containing promoters cells treated with siRNA against TAF4b (Fig. 4F). in vivo, ChIP was done with primers encompassing The role of integrin α6 in cell migration properties was the AP-1 site of the integrin α6 promoter schematic- verified in our cell system, as a reduction of integrin α6 le- ally represented in Fig. 6B. Interestingly, both c-Jun vels through siRNA resulted in an increase of the migration and TAF4b were found on the promoter of integrin α6 ability of Caco-2 (an increase of 50%), Caco-H (70%), and in Caco-2 and Caco-H cells whereas, surprisingly in HCT116 (90%) cells, as compared with cells treated with HCT116 cells, only TAF4b was bound (Fig. 6C and D). scrambled siRNA (siC; Fig. 5A). Investigating a possible Re-ChIP analysis of the c-Jun immunoprecipitate revealed coinvolvement of c-Jun in the regulation of integrin α6, that c-Jun and TAF4b simultaneously co-occupied the pro- we reduced c-Jun levels by siRNA and observed a decrease moter of integrin α6 in Caco-2 as well as in Caco-H cells

FIGURE 2. Expression levels of TAF4b and c-Jun in Caco-2 and Caco-H cells. A, total extracts from cells expressing mutated Ha-RAS (Caco-H1 and Caco-H2) were subjected to Western blotting analysis. B, real-time PCR analysis of c-Jun and TAF4b mRNA levels in the same cell lines. All data were normalized to GAPDH.

558 Mol Cancer Res; 8(4) April 2010 Molecular Cancer Research

TAF4b and c-Jun Regulate Cell Migration Properties

FIGURE 3. TAF4b influences cell migration. A, left, reduction of TAF4b (siTAF4b) and TAF4 (siTAF4) mRNA levels in Caco-H cells compared with nontarget siRNA (siC) and untreated cells (Control). Right, changes in Caco-H cell morphology after 72 h of TAF4b and TAF4 siRNA treatment. Arrows, protrusions formed after TAF4b siRNA treatment. Bottom, the percentage of cells with changed morphologies after TAF4b siRNA treatment compared with the total number of transfected cells. B, graphs showing the proliferation rate of untreated cells (Control), cells with nontarget siRNA (siControl) treatment, and cells with TAF4b (left) and TAF4 (right) siRNA treatment. The results are an average of three independent experiments. C, graph expressing the changes in the migratory ability of Caco-2, Caco-H, and HCT116 cells treated with siRNA, tested for their ability to migrate in response to a serum gradient. The results are an average of two independent experiments (*, P < 0.05; **, P < 0.01, as determined by Student's t test).

www.aacrjournals.org Mol Cancer Res; 8(4) April 2010 559

Kalogeropoulou et al.

FIGURE 4. TAF4b regulates the expression of integrins including integrin α6. A, B, and C, quantification of TAF4b, integrin α6 (ITGa6), integrin β4(ITGb4) and integrin β1 (ITGb1) mRNA levels via real-time PCR analysis, after treatment of Caco-2, Caco-H, and HCT116 cells with TAF4b siRNA (siTAF4b) compared with control nontarget siRNA-treated (siC) cells. D, left, real-time PCR analysis of integrin α6 mRNA levels in Caco-2, Caco-H (H1 and H2), and HCT116 cells. Right, immunofluorescence confocal imaging visualizing integrin α6 localization (green) in Caco-2 and Caco-H cells. Nuclei were stained with Hoechst dye. Merged images of nuclei and integrin α6 antibody are shown. E, immunofluorescence confocal imaging visualizing changes in signal intensity and localization of TAF4b and integrin α6 after treatment of cells with TAF4b-specific siRNA as compared with cells treated with control nontarget siRNA (siC). F, fluorescence-activated cell sorting analysis of integrin α6 expression levels in live Caco-H cells 48 h after transfection with control nontarget siRNA and TAF4b siRNA.

560 Mol Cancer Res; 8(4) April 2010 Molecular Cancer Research

TAF4b and c-Jun Regulate Cell Migration Properties

(Fig. 6E). In summary, TAF4b was found on the regula- the binding of c-Jun on the AP-1 site in Caco-H cells; tory AP-1 site of the integrin α6 promoter whereas its in- the anti-TAF4b antibody could not precipitate promoter terplay with c-Jun on this particular site, and only under a chromatinwhenc-Junwasknockeddown(Fig.7A,bot- specific cellular context, suggests a differential regulation tom).Onthecontrary,inCaco-2cells,TAF4bremained pattern. bound on the promoter even after the reduction of c-Jun To understand the importance of the cell context on the (siRNA c-Jun) protein levels. In the inverse situation in regulation pattern of integrin α6 by c-Jun and TAF4b, we which TAF4b levels were reduced by siRNA (Fig. 7B, knocked down c-Jun by siRNA in Caco-2 and Caco-H top), ChIP experiments showed that the binding of c-Jun cells (Fig. 7A), and followed the integrin α6 promoter oc- on the promoter of integrin α6 was independent of the pres- cupancy by TAF4b with ChIP analysis. Interestingly, the ence (siControl) or absence (siTAF4b) of TAF4b (bottom). binding of TAF4b on this promoter was dependent on This enforces the assumption that c-Jun controls the

FIGURE 5. Reduced levels of integrin α6 leads to higher rates of cell migration. A, left, graph expressing the changes in the migratory ability of each cell line after integrin α6 siRNA treatment, presented as an average of two independent experiments. Caco-2, Caco-H, and HCT116 cells were treated with control nontarget siRNA (siC) or integrin α6 siRNA. Right, reduction of integrin α6 (si-integrin α6) mRNA levels are shown compared with nontarget siRNA (siC; *, P < 0.05; **, P < 0.01, as determined by Student's t test). B, quantification of c-Jun and integrin α6 (ITGa6) mRNA levels in Caco-2, Caco-H, and HCT116 cells, after c-Jun siRNA (si-c-Jun) treatment. All data were normalized to GAPDH. C, fluorescence-activated cell sorting analysis of integrin α6 expression levels in live Caco-H cells 48 h after transfection with control nontarget siRNA and c-Jun siRNA.

www.aacrjournals.org Mol Cancer Res; 8(4) April 2010 561

Kalogeropoulou et al.

FIGURE 6. c-Jun and TAF4b bind on the AP-1 site of the promoter of integrin α6. A, left, chart showing the luciferase activity in Caco-2 cells transfected with 5xcoll-TRE-tata-luciferase plasmid or cotransfected with c-Jun (c-Jun-TRE-luc), TAF4b (TAF4b-TRE-luc), and TAF4 (TAF4-TRE-luc). Samples were prepared in triplicate and data are shown as an average of three independent experiments. All data are presented compared with control cells cotransfected with an empty luciferase vector (*, P < 0.05; **, P < 0.01, as determined by Student's t test). Right, total extracts analyzed by Western blotting, showing protein overexpression levels in Caco-2 cells. For the detection of overexpressed proteins, anti-Ha (TAF4b) and anti-His (c-Jun) antibodies were used, whereas for TAF4, an antibody against its protein was used. B, schema presenting the integrin α6 promoter close to its +1 site indicating the AP-1 site plus the region encompassed by the primers designed for the ChIP experiments; C, PCR end point; D, real-time PCR analysis of ChIP experiments on the AP-1 site of the integrin α6 promoter with chromatin extracted from Caco-2, Caco-H, and HCT116 cells. Immunoprecipitation with the indicated antibodies or, as a negative control, in the absence of any antibody (NoAb). D, results are normalized to inputs. E, Re-ChIP experiments with cross-linked chromatin from Caco-2 and Caco-H cells, using antibodies as indicated.

binding of TAF4b on the promoter of integrin α6in Cell Type–Specific AP-1 Complex Formation at the AP-1 Caco-H cells with an EMT-like phenotype, whereas in Site of the Integrin α6 Promoter Together with TAF4b Caco-2, this binding is independent of c-Jun. Therefore, To further clarify the AP-1 composition on the promoter a partial, cell type–specific c-Jun dependency of TAF4b- of integrin α6 in relation with TAF4b, we did Re-ChIP binding on this specific promoter is proposed. experiments in Caco-H cells. Interestingly, the integrin

562 Mol Cancer Res; 8(4) April 2010 Molecular Cancer Research

TAF4b and c-Jun Regulate Cell Migration Properties

FRA-1 was detected on the integrin α6promoterafter ChIP (Fig. 8B, top). Re-ChIP analysis (Fig. 8B, bottom) showed that in Caco-2 cells, in addition to c-Jun, FRA-1 was also present on the promoter. In the case of HCT116 cells, in which a c-Jun–independent binding of TAF4b was observed, FRA-1, ATF2, and c-Fos were identified on the promoter (Fig. 8C, top). Re-ChIP experiments further showed that TAF4b was able to interfere with FRA-1, ATF2, and c-Fos, but not FRA-2 (Fig. 8C, bottom). Be- cause c-Jun was not detected within the TAF4b/AP-1 family complex formed in HCT116 cells, we examined whether it could be substituted by another Jun family member on the integrin α6 promoter. Re-ChIP analysis showed that in HCT116 cells, JunB was detected in TAF4b-immunoprecipitated chromatin whereas in Caco- 2 and Caco-H cells, neither JunB nor JunD were present (Fig. 9A, top). Interestingly, JunB was not able to interact with TAF4b in the same cell line (Fig. 9A, bottom), indicating that the presence of both proteins, TAF4b and JunB, on the promoter of integrin α6 was promoter DNA–dependent. JunB siRNA treatment in HCT116

FIGURE 7. Binding of TAF4b on the promoter of target gene integrin α6is merely dependent on c-Jun. A and B, top, total protein extracts of Caco-H and Caco-2 cells analyzed by Western blotting, showing changes in protein levels of c-Jun, TAF4b, and integrin α6 after siRNA knockdown of c-Jun and TAF4b, respectively, Bottom, ChIP experiment done with the indicated antibodies under siControl treatment and c-Jun siRNA or TAF4b siRNA treatment, respectively, for 48 h.

α6 promoter was found to be occupied by FRA-2 and c-Fos in addition to c-Jun and TAF4b (Fig. 8A, i), indicating that c-Jun might dimerize with any of these proteins while in complex with TAF4b. To clarify the specificity of the complex formation between TAF4b and AP-1 fam- FIGURE 8. TAF4b forms a complex with different AP-1 family α ily members on specific promoters regulated by TAF4b, we members around the promoter of integrin 6, depending on the cell line. A, Re-ChIP experiments with Caco-H cells chromatin on the promoter followed with the same experiment on another promoter, of integrin α6 (i) and vimentin (ii). The first round of immunoprecipitation that of vimentin. Vimentin was not found to be regulated was done with anti-TAF4b, whereas the second immunoprecipitation by TAF4b in our system (Fig. 4E), and interestingly, was done with the indicated antibodies. B, top, ChIP experiment with TAF4b was also not found on its promoter together with Caco-2 cells. Immunoprecipitation with FRA-1 antibody. Bottom, Re-ChIP analysis in Caco-2 cells with antibodies as indicated. C, top, any AP-1 family member (Fig. 8A, ii). ChIP experiment in HCT116 cells. Binding of FRA-1, ATF2, and c-Fos On the other hand, in the Caco-2 cell line, in which on the AP-1 site of integrin α6 was tested. Bottom, Re-ChiP experiment TAF4b exhibited a c-Jun–independent binding, only with antibodies as indicated.

www.aacrjournals.org Mol Cancer Res; 8(4) April 2010 563

Kalogeropoulou et al.

FIGURE 9. JunB recruits TAF4b on the promoter of integrin α6 in HCT116 cells, replacing c-Jun. A, Re-ChIP experiments with cross-linked chromatin from Caco-2, Caco-H and HCT116 cells. The first round of immunoprecipitation was done with anti-TAF4b, whereas the second immunoprecipitation was done with the antibodies indicated (top). Western blotting analysis of immunoprecipitation with anti-JunB antibodies, using nuclear extracts from HCT116 cells. The samples were analyzed by Western blot (WB) with anti-JunB and anti-TAF4b antibodies (bottom). B, quantification of JunB and integrin α6 (ITGa6) mRNA levels in HCT116 (top) and Caco-H (bottom) cells, after JunB siRNA (siJunB) treatment as compared with cells treated with scrambled siRNA (siC). All data were normalized to GAPDH. C, top, total protein extracts analyzed by Western blotting, showing the change in protein levels of JunB and integrin α6 after JunB siRNA knockdown, in HCT116 and Caco-H cells. Bottom, ChIP experiment done with the indicated antibodies under siControl treatment and JunB siRNA treatment for 48 h in HCT116 and Caco-H cells. D, total extracts from Caco-2, Caco-H, and HCT116 cells subjected to Western blotting analysis using the antibodies indicated.

564 Mol Cancer Res; 8(4) April 2010 Molecular Cancer Research

TAF4b and c-Jun Regulate Cell Migration Properties

cells showed decreased levels of integrin α6 expression, whereas in Caco-H cells, under the same conditions, the expression of integrin α6 presented no significant changes (Fig. 9B). Therefore, to determine a possible JunB-dependence in the binding of TAF4b on the integrin α6-promoter in this particular cell line, HCT116, we followed with ChIP experiments after the knockdown of JunB by specific siRNA treatment (Fig. 9C, top). As a control, the same experiment was done in Caco-H cells in which JunB was not found on the promoter of integrin α6 together with TAF4b. Interestingly, the integrin α6 promoter occupancy in HCT116 cells by TAF4b showed a dependency on the presence of JunB because the anti- TAF4b antibody could not precipitate promoter chromatin after the exclusion of JunB (Fig. 9C, bottom). On the con- trary, in Caco-H cells, TAF4b remained on the promoter unaffected by the reduced JunB protein levels from siRNA. To assess both the preferential binding of specific AP-1 family members on the promoter of integrin α6 and their cell type–specific interplay with TAF4b, we analyzed the expression levels of particular AP-1 family members in total extracts of Caco-2, Caco-H, and HCT116 cells. As judged by Western blot analysis (Fig. 9D), the levels of these proteins were cell type–dependent. Interestingly, the expression levels of AP-1 factors c-Jun, FRA-2, and ATF2 correlated with the occupancy of integrin α6 promoter; whereas some of them, e.g., FRA-1, did not. The formation of complexes between AP-1 family members, together with TAF4b, might not be explained solely by the respective expression levels of each AP-1 member. FIGURE 10. Cell type–specific complex formation around the AP-1 site of α Validating the proposed preferential binding of Jun fam- integrin 6 evaluated by electrophoretic mobility shift assay analysis. Electrophoretic mobility shift assay was done using labeled probes ily members, together with TAF4b on the promoter of in- containing the AP-1 binding site of integrin α6. Probes were incubated tegrin α6, we analyzed the actual binding of protein in the presence (+) of Caco-2, Caco-H, or HCT116 nuclear extracts or in complexes on this exact DNA segment (AP-1 site) by gel the absence of any cell extract (−). For competition experiments, an shift assays using nuclear extracts from Caco-2, Caco-H, excess of the indicated unlabeled probe (competitor) was added. ETS 1.3 binding site was used as a nonspecific competitor. Free probe (F) and and HCT116 cells. Indeed, the presence of the integrin site-specific protein complexes (C) are indicated. α6 probe induced the specific binding of a protein complex in all cell lines (Fig. 10; lanes 5, 6, and 7). Interest- ingly, the DNA-binding activity in each cell line was relevant to the expression levels of integrin α6 in the re- Discussion spective cell line, as shown in Fig. 5C. Caco-H and HCT116 cell extracts (lanes 6 and 7) showed stronger pro- For many years, it had been proposed that TAFs were tein complex signals as compared with Caco-2 (lane 5). To transmitters of information between activators and the core exclude any off-target effects, we added an excess of non- transcriptional machinery. Notably, it has been shown that radiolabeled integrin α6 probe (lane 4), which almost abol- individual TAFs are required for the expression of only a ished the protein complex formation on the AP-1 site of specific subset of genes (35-38), and that the TFIID, or integrin α6, whereas with other nonspecific competitors any of its other forms, was recruited in core promoters (lanes 2 and 3), the complex formations on these sites re- by a direct interaction between TAFs and their gene- mained intact. In agreement, a nonspecific probe (cold specific activators (39). Given that TAF4b target pro- ETS 1.3) did not affect the intensity of the signal (lane moter selectivity is enhanced by activators such as c-Jun 2) whereas a cold vimentin probe (Vim) containing an and Sp-1 (12), we propose a synergistic function of the AP-1 site competed with the integrin α6 probe (lane 3). two factors, c-Jun and TAF4b, through their interaction Moreover, an integrin α6–mutated (iα6mut,lane8) in which TAF4b is the coactivator of AP-1 during regu- probe, bearing three point mutations, failed to build the lation of AP-1 target genes (Fig. 11). Importantly, because same protein complex as the wild-type AP-1 site of integrin among all the human TAFs, only TAF4b was found to α6. These results indeed validate the protein complex for- interact with c-Jun, we cannot exclude the possibility of mation around the AP-1 binding site of the integrin α6 this interaction taking place independently of the TFIID promoter. complex.

www.aacrjournals.org Mol Cancer Res; 8(4) April 2010 565

Kalogeropoulou et al.

FIGURE 11. Model of TAF4b/AP-1 complex function on target genes, depending on their cellular environment. According to its cellular environment, TAF4b forms distinct complexes with AP-1 family members to drive transcriptional activation. A, activator (c-Jun) plus coactivator (TAF4b)–dependent transcriptional activation on the promoter of integrin α6 in the model of Caco-H cells. TAF4b recruitment to the AP-1 site of the promoter and subsequent activation of transcription is dependent on c-Jun. B, activation of transcription by TAF4b and FRA-1. c-Jun is also present in the promoter but recruitment of TAF4b on the AP-1 site is not solely dependent on c-Jun itself. C, transcriptional activation of target gene by TAF4b and Jun family member JunB in HCT116 cells. JunB, which is overexpressed in these cells, takes over the transactivation role of c-Jun.

Interestingly, TAF12, which heterodimerizes with TAF4 AP-1 Family Members Share Transcriptional Regulation or TAF4b, has also been shown to directly interact with of Target Genes through Interaction with TAF4b AP-1 family member ATF7, controlling transcription Based on our observations, the modulation of AP-1 com- (40). In the present study, we suggest that TAF4b func- position in response to external signals, possibly by exchang- tions as a coactivator of integrin α6 transcription in con- ing the most abundant AP-1 family member in each cell cert with activator c-Jun or, depending on the cell type, type, serves as a regulatory mechanism triggering the expres- with other AP-1 family members. We propose that TAF4b sion of specific genes. Indeed, we have shown that in the together with AP-1 factors induces preinitiation complex integrin α6 promoter, c-Jun is not the only Jun family mem- formation and transcriptional regulation of certain genes ber occupying the AP-1 site together with TAF4b but might containing an AP-1 binding site (Fig. 8). as well be replaced by JunB, for example, within HCT116 The coactivator domain of TAF4 and TAF4b is not con- cells. Other AP-1 family members were found on this site as served, leading to the hypothesis of an independent se- well, probably building dimers in the absence or presence of quence-specific coactivator function. Our analysis, using c-Jun and in a cell type–dependent mechanism, to some ex- luciferase reporter constructs transfected to colorectal can- tent, in an expression level–proportional manner (Fig. 8). cer cells, supports that TAF4b, but not TAF4, acts as a Because the protein levels of transcription factors do not per- coactivator on specific AP-1 targets. Furthermore, in our fectly correlate with the pattern of occupancy of the integrin study, we observed that an intracellular reduction of α6 promoter, other mechanisms that fine-tune the affinity TAF4b might be compensated by induced expression levels of particular AP-1 family members on the cognate cis ele- of TAF4 (Supplementary Data), reinforcing the previous ment could be considered. For instance, the interaction with assumption that TAF4 was limiting the TAF4b-containing TAF4b or the existence of particular posttranslational mod- TFIID complexes present in cells (36). Interestingly, TAF4 ifications could result in DNA-affinity alteration. In this and TAF4b, through their competitive equilibrium in vein, it is well known that the mutational profile of each cell TFIID, have been shown to regulate the expression of line influences signaling pathways such as JNK and mito- genes involved in cell proliferation and transforming gen-activated protein kinase, which are crucial for AP-1 reg- growth factor-β signaling, known to drive tumor cells to ulation. Notably, the HCT116 cell line bears, among others, EMT transformation (41, 42). This underlines the impor- endogenous mutated forms of K-RAS and PIK3CA onco- tance of further analyzing the gene- and signal-specific genes, whereas the Caco-H cell line overexpresses a mutated coactivator functions of both factors. Ha-RAS oncogene leading to high levels of JNK activity

566 Mol Cancer Res; 8(4) April 2010 Molecular Cancer Research

TAF4b and c-Jun Regulate Cell Migration Properties

(19). Our study illustrates the significance of screening dif- known to play a critical role in a number of cellular func- ferent cell types as a way to link genetic alterations to partic- tions including cell migration and differentiation (47). ular patterns of gene regulation. Both integrins have been found elevated in several types Jun proteins have diverse expression profiles and biolog- of carcinomas, whereas increased expression levels correlate ical functions, even though they share high sequence ho- with invasive phenotypes and EMT (48, 49). Integrin α6is mologies. Although, c-Jun and JunB may induce opposite a controversial member of the integrin family because it has effects (43), they also have gene targets in common (44). been shown to have a dual role depending on the mutation- Interestingly, JunB has been suggested to substitute c-Jun al oncogenic profile of the tumor tissue and its stage (50). during mouse development and cell proliferation when Interestingly, in human mammary cancer and in mouse c-Jun is depleted (45), further supporting our suggestion keratinocytes, reduced α6 expression has been linked to in- that AP-1 family members take their promoter-specific creased cell migration and metastasis (51, 52). On the onset interaction with TAF4b into their own hands and follow of cell migration, cells must detach from the basal mem- with regulation of transcription. brane, in part by reducing the expression of integrin α6, which is responsible for detachment and migration. On TAF4b is Linked to Cell Migration Mechanisms and EMT the other hand, when gaining a more mesenchymal pheno- The involvement of other TAFs in cancer and/or metas- type, integrin α6 expression must again be upregulated to tasis has already been brought into light (7, 46), however, invade the basal membrane. Nevertheless, under conditions this is the first report linking TAF4b itself with the regula- of induced migration ability (e.g., after TAF4b knock- tion of tumor cell migration. Herewith, we provide evidence down), integrin α6 expression must be downregulated so that TAF4b is a suppressor of cell migration regulating, in that migration could begin. Accordingly, integrin α6ex- concert with c-Jun, the expression of genes playing a pivotal pression was reduced in tumors formed after the injection role in EMT. Accordingly, in a microarray analysis done in of Caco-H cells in severe combined immunodeficiency granulosa cells stably overexpressing TAF4b, a number of mice, in comparison with cultured cells, supporting its AP-1 target genes were upregulated (13), including the role in invasion mechanisms (28). EMTmarker vimentin. In our hands, the expression of this In conclusion, we report a cell type–specific initiation of gene was unaffected by the alteration of TAF4b levels in transcription controlled by TAF4b together with variant Caco-H cells, possibly due to an already highly activated AP-1 family members in a promoter-dependent manner, state of this promoter in the EMT cells. Indeed, in these emphasizing the importance of deciphering the indepen- particular cells, in contrast with the epithelial Caco-2 cells, dent roles of specific TFIID subunits as cofactors that gov- the vimentin gene was found to be predominantly regulated ern transcription. by FRA-1 (19), which did not interact with TAF4b in our experiments, suggesting a cell type–specific and TAF4b- Disclosure of Potential Conflicts of Interest independent mechanism regulated by AP-1 family member selectivity. On the other hand, TAF4b was shown to regulate No potential conflicts of interest were disclosed. integrin α6 in different cell lines: a colon adenocarcinoma cell line, its derivative Caco-H cell line constitutively over- Grant Support expressing Ha-RAS, and in parallel, in an established colon Marie Curie Fellowship and EU Marie Curie Research Training Network “TAF- cell line that has gained some EMT characteristics. Interest- Chromatin” grant MRTN-CT-2004 504228 (L. Tora, A. Pintzas, and R. Dikstein). ingly, in another study (36), integrin α6 was found to be The costs of publication of this article were defrayed in part by the payment of upregulated in mouse fibroblasts after knockdown of page charges. This article must therefore be hereby marked advertisement in TAF4 and its proven replacement by TAF4b. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. α β The integrin subunits 6and 4 together form the Received 04/15/2009; revised 03/02/2010; accepted 03/03/2010; published hemidesmosomal α6β4 integrin, a transmembrane receptor OnlineFirst 03/30/2010.

References 1. Bell B, Tora L. Regulation of gene expression by multiple forms of and T lymphocytes in TAFII105 dominant-negative transgenic mice is TFIID and other novel TAFII-containing complexes. Exp Cell Res linked to nuclear factor-κB. J Biol Chem 2002;277:17821–9. 1999;246:11–9. 6. Lu H, Levine AJ. Human TAFII31 protein is a transcriptional 2. Demeny MA, Soutoglou E, Nagy Z, et al. Identification of a small TAF coactivator of the p53 protein. Proc Natl Acad Sci U S A 1995;92: complex and its role in the assembly of TAF-containing complexes. 5154–8. PLoS ONE 2007;2:e316. 7. Voulgari A, Voskou S, Tora L, et al. TATA box-binding protein- 3. Wassarman DA, Aoyagi N, Pile LA, Schlag EM. TAF250 is required associated factor 12 is important for RAS-induced transformation for multiple developmental events in Drosophila. Proc Natl Acad Sci properties of colorectal cancer cells. Mol Cancer Res 2008;6: U S A 2000;97:1154–9. 1071–83. 4. Metzger D, Scheer E, Soldatov A, Tora L. Mammalian TAF(II)30 is 8. Voronina E, Lovasco LA, Gyuris A, Baumgartner RA, Parlow AF, required for cell cycle progression and specific cellular differentiation Freiman RN. Ovarian granulosa cell survival and proliferation re- programmes. EMBO J 1999;18:4823–34. quires the gonad-selective TFIID subunit TAF4b. Dev Biol 2007; 5. Silkov A, Wolstein O, Shachar I, Dikstein R. Enhanced apoptosis of B 303:715–26.

www.aacrjournals.org Mol Cancer Res; 8(4) April 2010 567

Kalogeropoulou et al.

9. Dikstein R, Zhou S, Tjian R. Human TAFII 105 is a cell type-specific genetic programs of cellular transformation. Genes Dev 1998;12: TFIID subunit related to hTAFII130. Cell 1996;87:137–46. 1227–39. 10. Rashevsky-Finkel A, Silkov A, Dikstein R. A composite nuclear 32. Angel P, Hattori K, Smeal T, Karin M. Oncogene jun encodes a export signal in the TBP-associated factor TAFII105. J Biol Chem sequence-specific trans-activator similar to AP-1. Cell 1988;55: 2001;276:44963–9. 875–85. 11. Shao H, Revach M, Moshonov S, et al. Core promoter binding by 33. Gangloff YG, Werten S, Romier C, et al. The human TFIID compo- histone-like TAF complexes. Mol Cell Biol 2005;25:206–19. nents TAF(II)135 and TAF(II)20 and the yeast SAGA components 12. Liu WL, Coleman RA, Grob P, et al. Structural changes in TAF4b- ADA1 and TAF(II)68 heterodimerize to form histone-like pairs. Mol TFIID correlate with promoter selectivity. Mol Cell 2008;29:81–91. Cell Biol 2000;20:340–51. 13. Geles KG, Freiman RN, Liu WL, Zheng S, Voronina E, Tjian R. Cell- 34. Nishida K, Kitazawa R, Mizuno K, Maeda S, Kitazawa S. Identi- type-selective induction of c-jun by TAF4b directs ovarian-specific fication of regulatory elements of human α6 integrin subunit gene. transcription networks. Proc Natl Acad Sci U S A 2006;103:2594–9. Biochem Biophys Res Commun 1997;241:258–63. 14. Bannister AJ, Oehler T, Wilhelm D, Angel P, Kouzarides T. 35. Lee TI, Causton HC, Holstege FC, et al. Redundant roles for the Stimulation of c-Jun activity by CBP: c-Jun residues Ser63/73 TFIID and SAGA complexes in global transcription. Nature 2000; are required for CBP induced stimulation in vivo and CBP binding 405:701–4. in vitro. Oncogene 1995;11:2509–14. 36. Mengus G, Fadloun A, Kobi D, et al. TAF4 inactivation in embryonic 15. Munz C, Psichari E, Mandilis D, et al. TAF7 (TAFII55) plays a role in fibroblasts activates TGF β signalling and autocrine growth. EMBO J the transcription activation by c-Jun. J Biol Chem 2003;278:21510–6. 2005;24:2753–67. 16. Halazonetis TD, Georgopoulos K, Greenberg ME, Leder P. c-Jun 37. Fadloun A, Kobi D, Pointud JC, et al. The TFIID subunit TAF4 dimerizes with itself and with c-Fos, forming complexes of different regulates keratinocyte proliferation and has cell-autonomous DNA binding affinities. Cell 1988;55:917–24. and non-cell-autonomous tumour suppressor activity in mouse 17. Hess J, Angel P, Schorpp-Kistner M. AP-1 subunits: quarrel and epidermis. Development 2007;134:2947–58. harmony among siblings. J Cell Sci 2004;117:5965–73. 38. Tatarakis A, Margaritis T, Martinez-Jimenez CP, et al. Dominant and 18. Zoumpourlis V, Papassava P, Linardopoulos S, Gillespie D, redundant functions of TFIID involved in the regulation of hepatic Balmain A, Pintzas A. High levels of phosphorylated c-Jun, Fra-1, genes. Mol Cell 2008;31:531–43. Fra-2 and ATF-2 proteins correlate with malignant phenotypes in 39. Mencia M, Moqtaderi Z, Geisberg JV, Kuras L, Struhl K. Activator- the multistage mouse skin carcinogenesis model. Oncogene 2000; specific recruitment of TFIID and regulation of ribosomal protein 19:4011–21. genes in yeast. Mol Cell 2002;9:823–33. 19. Andreolas C, Kalogeropoulou M, Voulgari A, Pintzas A. Fra-1 40. Hamard PJ, Dalbies-Tran R, Hauss C, Davidson I, Kedinger C, regulates vimentin during Ha-RAS-induced epithelial mesenchymal Chatton B. A functional interaction between ATF7 and TAF12 that transition in human colon carcinoma cells. Int J Cancer 2008;122: is modulated by TAF4. Oncogene 2005;24:3472–83. 1745–56. 41. Davidson I, Kobi D, Fadloun A, Mengus G. New insights into TAFs 20. Mechta F, Lallemand D, Pfarr CM, Yaniv M. Transformation by as regulators of cell cycle and signaling pathways. Cell Cycle 2005;4: ras modifies AP1 composition and activity. Oncogene 1997;14: 1486–90. 837–47. 42. Pardali K, Moustakas A. Actions of TGF-β as tumor suppressor and 21. Bos TJ, Margiotta P, Bush L, Wasilenko W. Enhanced cell motility pro-metastatic factor in human cancer. Biochim Biophys Acta 2007; and invasion of chicken embryo fibroblasts in response to Jun 1775:21–62. over-expression. Int J Cancer 1999;81:404–10. 43. Shaulian E, Karin M. AP-1 as a regulator of cell life and death. Nat 22. Smeal T, Binetruy B, Mercola DA, Birrer M, Karin M. Oncogenic and Cell Biol 2002;4:E131–6. transcriptional cooperation with Ha-Ras requires phosphorylation of 44. Leaner VD, Kinoshita I, Birrer MJ. AP-1 complexes containing cJun c-Jun on serines 63 and 73. Nature 1991;354:494–6. and JunB cause cellular transformation of Rat1a fibroblasts and 23. Johnson R, Spiegelman B, Hanahan D, Wisdom R. Cellular transfor- share transcriptional targets. Oncogene 2003;22:5619–29. mation and malignancy induced by ras require c-jun. Mol Cell Biol 45. Passegue E, Jochum W, Behrens A, Ricci R, Wagner EF. JunB 1996;16:4504–11. can substitute for Jun in mouse development and cell proliferation. 24. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigen- Nat Genet 2002;30:158–66. esis. Cell 1990;61:759–67. 46. Nagy Z, Tora L. Distinct GCN5/PCAF-containing complexes function 25. Grunert S, Jechlinger M, Beug H. Diverse cellular and molecular as co-activators and are involved in transcription factor and global mechanisms contribute to epithelial plasticity and metastasis. Nat histone acetylation. Oncogene 2007;26:5341–57. Rev Mol Cell Biol 2003;4:657–65. 47. Watt FM. Role of integrins in regulating epidermal adhesion, growth 26. Huber MA, Kraut N, Beug H. Molecular requirements for epithelial- and differentiation. EMBO J 2002;21:3919–26. mesenchymal transition during tumor progression. Curr Opin Cell 48. Dajee M, Tarutani M, Deng H, Cai T, Khavari PA. Epidermal Ras Biol 2005;17:548–58. blockade demonstrates spatially localized Ras promotion of prolifer- 27. Janda E, Lehmann K, Killisch I, et al. Ras and TGFβ cooperatively ation and inhibition of differentiation. Oncogene 2002;21:1527–38. regulate epithelial cell plasticity and metastasis: dissection of Ras 49. Cruz-Monserrate Z, O'Connor KL. Integrin α6β4 promotes migration, signaling pathways. J Cell Biol 2002;156:299–313. invasion through Tiam1 upregulation, and subsequent Rac activa- 28. Roberts ML, Drosopoulos KG, Vasileiou I, et al. Microarray analysis tion. Neoplasia 2008;10:408–17. of the differential transformation mediated by Kirsten and Harvey 50. Raymond K, Kreft M, Song JY, Janssen H, Sonnenberg A. Dual Ras oncogenes in a human colorectal adenocarcinoma cell line. role of α6β4 integrin in epidermal tumor growth: tumor-suppressive Int J Cancer 2006;118:616–27. versus tumor-promoting function. Mol Biol Cell 2007;18:4210–21. 29. Schreiber E, Matthias P, Muller MM, Schaffner W. Rapid detection 51. Natali PG, Nicotra MR, Botti C, Mottolese M, Bigotti A, Segatto O. of octamer binding proteins with ‘mini-extracts’, prepared from a Changes in expression of α6/β4 integrin heterodimer in primary and small number of cells. Nucleic Acids Res 1989;17:6419. metastatic breast cancer. Br J Cancer 1992;66:318–22. 30. Chen C, Okayama H. High-efficiency transformation of mammalian 52. Rodius S, Indra G, Thibault C, Pfister V, Georges-Labouesse E. cells by plasmid DNA. Mol Cell Biol 1987;7:2745–52. Loss of α6 integrins in keratinocytes leads to an increase in TGFβ 31. van Dam H, Huguier S, Kooistra K, et al. Autocrine growth and and AP1 signaling and in expression of differentiation genes. J Cell anchorage independence: two complementing Jun-controlled Physiol 2007;212:439–49.

568 Mol Cancer Res; 8(4) April 2010 Molecular Cancer Research

TAF4b and Jun/Activating Protein-1 Collaborate to Regulate the Expression of Integrin α6 and Cancer Cell Migration Properties

Margarita Kalogeropoulou, Angeliki Voulgari, Vassiliki Kostourou, et al.

Mol Cancer Res 2010;8:554-568. Published OnlineFirst March 30, 2010.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-09-0159

Supplementary Access the most recent supplemental material at: Material http://mcr.aacrjournals.org/content/suppl/2010/03/29/1541-7786.MCR-09-0159.DC1

Cited articles This article cites 52 articles, 18 of which you can access for free at: http://mcr.aacrjournals.org/content/8/4/554.full#ref-list-1

Citing articles This article has been cited by 4 HighWire-hosted articles. Access the articles at: http://mcr.aacrjournals.org/content/8/4/554.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mcr.aacrjournals.org/content/8/4/554. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.