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

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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) fam- ily members participate in oncogenic transformation via gene regulation. Utilizing immunoprecipitation of en- dogenous 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 chro- matin 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 tran- scription 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 acti- vators 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 Downloaded from mcr.aacrjournals.org on September 27, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst March 30, 2010; DOI: 10.1158/1541-7786.MCR-09-0159 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 immuno- precipitation 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,
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