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Open Full Page Molecular Cancer Signaling and Regulation Research Expression Regulation of the Metastasis-Promoting Protein InsP3-Kinase-A in Tumor Cells Lydia Chang1, Heidi Schwarzenbach2,Sonke€ Meyer-Staeckling2, Burkard Brandt2, Georg W. Mayr1, Joachim M. Weitzel3, and Sabine Windhorst1 Abstract – Under physiologic conditions, the inositol-1,4,5-trisphosphate (InsP3)-metabolizing, F-actin bundling InsP3- kinase-A (ITPKA) is expressed only in neurons. Tumor cells that have gained the ability to express ITPKA show an increased metastatic potential due to the migration-promoting properties of ITPKA. Here we investigated the mechanism how tumor cells have gained the ability to reexpress ITPKA by using a breast cancer cell line (T47D) with no expression and a lung carcinoma cell line (H1299) with ectopic ITPKA expression. Cloning of a 1,250-bp ITPKA promoter fragment revealed that methylation of CpG islands was reduced in H1299 as compared with T47D cells, but DNA demethylation did not alter the expression of ITPKA. Instead, we showed that the repressor-element-1–silencing transcription factor (REST)/neuron-restrictive silencer factor (NRSF), which suppresses expression of neuronal genes in nonneuronal tissues, regulates expression of ITPKA. Knockdown of REST/NRSF induced expression of ITPKA in T47D cells, whereas its overexpression in H1299 cells strongly reduced the level of ITPKA. In T47D cells, REST/NRSF was bound to the RE-1 site of the ITPKA promoter and strongly reduced its activity. In H1299 cells, in contrast, expressing comparable REST/NRSF levels as T47D cells, REST/NRSF only slightly reduced ITPKA promoter activity. This reduced suppressor activity most likely results from expression of a dominant-negative isoform of REST/NRSF, REST4, which impairs binding of REST/NRSF to the RE-1 site. Thus, ITPKA may belong to the neuronal metastasis-promoting proteins whose ectopic reexpression in tumor cells is associated with impaired REST/NRSF activity. Mol Cancer Res; 9(4); 497–506. Ó2011 AACR. Introduction mers, ITPKA bundles actin filaments, resulting in increased size and/or number of dendritic spines (4, 5). In tumor cells InsP3-kinase-A (ITPKA) activity phosphorylates the cal- that have gained the ability to express ITPKA, the F-actin cium-mobilizing second messenger D-myo-inositol-1,4,5- bundling activity of ITPKA, and its interaction with the F- trisphosphate [Ins(1,4,5)P ]toD-myo-inositol-1,3,4,5-tet- actin cross-linking protein filamin C, induces the formation 3 – rakisphosphate [Ins(1,3,4,5)P4] and thus is involved in the of new filopodia and lamellipodia (6 8). As formation of regulation of calcium signaling (1). Under physiologic these cellular protrusions is a prerequisite for cells to conditions, ITPKA is expressed only in neurons of the migrate, the actin-modulating activity of ITPKA increases hippocampus, neocortex, and cerebellum (GeneAtlas, the migratory and the metastatic potential of tumor cells (6). V133A gcma, BioGPS; ref. 2) where it accumulates in In addition, ITPKA promotes migration by its ITPK dendritic spines via its association with F-actin (3). Because activity mediating the induction of store-operated calcium of this association and its characteristic to form homodim- entry and with this the stimulation of calcium-dependent actin-modulating proteins (6). Current studies on lung cancer patients show that adenocarcinoma cells express high Authors' Affiliations: 1Institut fur€ Biochemie und Molekularbiologie I: Zellulare€ Signaltransduktion; 2Institut fur€ Tumorbiologie, Universitatsklini-€ levels of ITPKA in the primary tumors and in metastasis, kum Hamburg-Eppendorf, Hamburg; and 3Leibniz-Institut fur€ Nutztierbio- whereas ITPKA expression was mainly found in metastasis logie, Dummerstorf, Dummerstorf, Mecklenburg-Vorpommern, Germany in small cell and squamous carcinoma (7). Furthermore, it Note: Supplementary data for this article are available at Molecular Cancer has been shown that only particular tumor cell lines could Research Online (http://mcr.aacrjournals.org/). express ITPKA (6, 7). However, the mechanism underlying Corresponding Author: Sabine Windhorst, Institut fur€ Biochemie this tumor cell and stage-specific regulation of ITPKA und Molekularbiologie I: Zellulare€ Signaltransduktion, Universitatskli-€ nikum Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, expression has not been elucidated yet. Germany. Phone: 49-40-7410-56341; Fax: 49-40-7410-56818. E-mail: Because the knowledge of regulation of ITPKA expres- [email protected] sion in tumor cells may provide the possibility to inhibit doi: 10.1158/1541-7786.MCR-10-0556 reexpression of ITPKA, here we examined the mechanism Ó2011 American Association for Cancer Research. underlying expression regulation of ITPKA in tumor cells. www.aacrjournals.org 497 Downloaded from mcr.aacrjournals.org on September 27, 2021. © 2011 American Association for Cancer Research. Chang et al. Materials and Methods Transient transfections pCMV6-vector (as a control) and pCMV6-XL4 REST Cell culture (for REST overexpression) were gifts from Pranela Ramesh- NCI-H1299 (H1299) cells were kindly provided by war (Newark, NJ). Transfections were done by using Cagatay Gunes€ (Hamburg, Germany) and T47D cells by Lipofectamine reagent (Invitrogen), according to the man- Udo Schumacher (Hamburg, Germany). T47D cells are ufacturer's instructions. Transfected cells were cultured for mammary epithelial gland ductal carcinoma cells derived 48 hours, lysed with M-PER Buffer (Thermo Scientific), or from pleural effusion. They are estrogen receptor positive fixed with 3% paraformaldehyde. and express mutated p53 gene. MCF-7 cells are mammary epithelial gland adenocarcinoma cells derived from pleural Western blot effusion. They are estrogen receptor positive but lack To detect proteins, standard SDS-PAGE and immuno- expression of Her2/neu. NCI-H1299 cells are epithelial blot techniques were applied. Blots were developed using non–small cell lung cancer (NSCLC) cells derived from a the ECL Plus system (GE Healthcare), according to the lymph node metastasis. They have a homozygous partial manufacturer's instructions. Equal gel loading was exam- deletion of the p53 gene and lack expression of p53 (source: ined by glyceraldehyde 3-phosphate dehydrogenase American Tissue Culture Collection). The cell line NCI- (GAPDH) expression. Antibodies were purchased from H1299 was cultured in DMEM, and T47D and MCF-7 Santa Cruz: GAPDH (Sc69778), cyclin A (Sc-596), and cells were grown in RPMI; both media were supplemented ITPKA (Sc69778). Specifity of the ITPKA antibody was with 10% (v/v) fetal calf serum, 3.97 mmol/L L-glutamine, evaluated in a former study (7). In addition, brain lysates of 100 mg/mL streptomycin, and 100 U/mL penicillin. Media ITPKA wild-type and knockout mice were analyzed by were purchased from Invitrogen. Western blotting (Supplementary Fig. S1). Cloning, transfections, and luciferase assays RNA preparation, RT-PCR, and real-time PCR To amplify DNA fragments upstream of the ITPKA Total RNA was prepared from cell lines using the gene, standard PCRs were carried out in the presence of NucleoSpin RNAII Kit (Machery & Nagel). The recovery sequence-specific primers surrounded by XhoI/HindIII of RNA was quantified spectrophotometrically. One micro- sites for directional cloning. The resulting DNA frag- gram of RNA was synthesized into cDNA with Superscript ments were inserted into the reporter firefly luciferase III, reverse transcriptase, and oligo(dT) primers (all from vector pGL3-basic vector (Promega), in which the back- Invitrogen), according to the manufacturer's instructions. bone was previously digested with XhoI/HindIII, to The cDNA was used as template for semiquantitative or delete the coding sequence for the SV40 promoter. Thus, real-time PCR with gene-specific primers, according to the the ITPKA promoter fragments were cloned into a manufacturer's instructions (Roche). Primer sequences used promoter-less vector. The same promoter-less vector were as follows: served as a negative control to measure background Hsc70: upstream (GCT GCT GCT ATT GCT TAC activity. The altered pGL3 constructs were confirmed GGC); downstream (TGC TGG AAGGAG GGT ACG by sequencing. Transfections of H1299 and T47D cells CT) were carried out by using Lipofectamine reagent (Invi- ITPKA: upstream (AGC TGC AGG ACC TGC TCG trogen), according to the manufacturer's instructions. AT); downstream (TCC GTG GGA GCT TCA GGA After transfection, cells were cultured for 16 hours in TC) serum-free medium and lysed with Lysis Buffer (Pro- REST1: upstream (GCT ACA GTT ATG GCC ACC mega). Luciferase activity was determined with the Dual- CAG GTG AT); downstream (GGC TTC TCA CCC Glo Luciferase Assay Reagent (Promega), according to ATC TAG ATC CAC T) the manufacturer's instructions by using a Berthold REST4: upstream (CTACAT GGC ACA CCT GAA luminometer (Berthold). Luciferase activity values were GCA CAC); downstream (GGC TTT CAC CCA TCT corrected by internal normalization performing cotrans- AGA TCA CAC T) fection of the pGL4.74 vector (Promega) containing Cyclin D: upstream (CCG TCC ATG CGG AAG Renilla luciferase. Luciferase assays were carried out in ATC), downstream (GAA GAC CTC CTC CTC duplicate, and each experiment of transfection was per- GCA CT) formed at least 3 times. The coding region of REST4 was amplified by PCR from pCMV6-XL4 REST (see the following text), using the Determination of mRNA and protein half-life oligonucleotides GGT ACC ATG GCC ACC CAG For examination of mRNA half-life, H1299 cells were GTA ATG GGG (sense) and GGATCC TCA CCC seeded in 24-well plates and grown to 80% confluence. AAC TAG ATC ACA ACC TGA ATG AGT ACG Then, the cells were treated with 10 mg/mL actinomycin for CAT ATG (antisense) surrounded by restriction sites for 0, 1, 3, 6, 16, and 24 hours. The mRNA level was KpnI and BamHI for directional cloning into the plasmid determined by real-time PCR as described earlier. For each vector pEGFP-C1 (Clontech TakaraBio) to generate sample, experiments were done in duplicates. To analyze EGFP–REST4. protein half-life, cells grown in full medium were treated 498 Mol Cancer Res; 9(4) April 2011 Molecular Cancer Research Downloaded from mcr.aacrjournals.org on September 27, 2021.
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