A single mutation in Securin induces chromosomal instability and enhances cell invasion

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published by Elsevier in: EUROPEAN JOURNAL OF CANCER on 2013, available at: https://doi.org/10.1016/j.ejca.2012.06.024” 1 A Single Mutation in Securin Induces Chromosomal Instability and Enhances Cell Invasion Mar Mora-Santos1, Carolina Castilla2, Joaquín Herrero-Ruiz1, Servando Giráldez1, M. Cristina Limón-Mortés1, Carmen Sáez2, Miguel Á. Japón2, Maria Tortolero1 and Francisco Romero1* 1 Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla. Apartado de correos 1095. 41080-Sevilla, Spain. 2 Instituto de Biomedicina de Sevilla (IBIS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla and Departamento de Anatomía Patológica, Hospital Universitario Virgen del Rocío, 41013 Sevilla, Spain. * Corresponding author. Tel: 34-954557119. Fax: 34-954557830. E-mail: [email protected] *Manuscript 2 ABSTRACT Pituitary tumor transforming gene (pttg1) encodes Securin, a protein involved in the inhibition of sister chromatid separation binding to Separase until the onset of anaphase. Separase is a cysteine-protease that degrades cohesin to segregate the sister chromatids to opposite poles of the cell. The amount of Securin is strongly regulated because it should allow Separase activation when is degraded by the anaphase promoting complex/cyclosome, should arrest the cell cycle after DNA damage, when is degraded through SKP1-CUL1-ETrCP ubiquitin ligase, and its overexpression induces tumor formation and correlates with metastasis in multiple tumors. Securin is a phosphoprotein that contains 32 potentially phosphorylatable residues. We mutated and analyzed most of them, and found a single mutant, hSecT60A, that showed enhanced oncogenic properties. Our fluorescence activated cell sorting analysis, fluorescence in situ hybridization assays, tumor cell migration and invasion experiments and gene expression by microarrays analysis clearly involved hSecT60A in chromosomal instability and cell invasion. These results show, for the first time, that a single mutation in pttg1 is sufficient to trigger the oncogenic properties of Securin. The finding of this point mutation in patients might be used as an effective strategy for early detection of cancer. Keywords: Securin, pttg, cell invasiveness, tumorigenesis, chromosomal instability, pituitary tumor, human mutation. 3 INTRODUCTION Securin is a protein widely distributed in all eukaryotic organisms that is involved in control of the metaphase-to-anaphase transition (1-4). Securin inhibits the cysteine protease Separase until the onset of anaphase, in which it is ubiquitinated by the anaphase promoting complex/cyclosome (APC/C) E3 ubiquitin ligase and degraded via proteasome. Then, activated Separase cleaves cohesin and releases sister chromatids (5- 7). Moreover, there is much evidence that Securin is important for cellular responses to genotoxic stresses, delaying the onset of anaphase to allow repair before sister chromatids are segregated from each other (8-10). The pttg1 gene, first isolated from rat pituitary cells and a human T lymphocyte cell line, encodes a protein that was identified as a Securin homolog (4, 11, 12). Human Securin is a protein overproduced in a wide variety of tumor cell lines and human cancers (11, 13-16). hSecurin shows the ability to induce cellular transformation in vitro and in vivo. In fact, introduction of hSecurin into HEK293 cells increased cell proliferation, anchorage-independent growth in soft agar and formation of tumors after subcutaneous injection of nu/nu mice (17). Furthermore, hSecurin abundance is a molecular marker for invasiveness in hormone-secreting pituitary tumors, is associated with invasion of surrounding lymph nodes, and its overexpression correlates with metastasis in multiple tumors (16, 18, 19). Securin also plays a role in transcriptional regulation. The Securin protein structure and some ex vivo experiments prompted us to suggest that Securin might have a role in the transcriptional activation of other genes (11). Later, it has been shown that several genes involved in cellular growth and angiogenesis are transactivated by Securin. In this way, Securin induction in HeLa cells results in increased cell proliferation through c-Myc activation (20). On the other hand, 4 both expression and secretion of FGF-2, and expression of VEGF are induced by Securin (21-23). Mutation of proline-rich motifs of murine Securin resulted in the loss of in vitro transforming activity, the in vivo tumor-inducing potential of Securin and also abrogated its transactivating ability, suggesting that the transactivation and transforming abilities of Securin are associated (17, 23, 24). hSecurin is a cell cycle-dependent protein that is phosphorylated at serine 165 by CDK1 (25). Mutation of this serine did not prevent Securin degradation during mitosis, inhibition of chromatid separation or FGF-2 induction (26). However, hSecurin must be phosphorylated on other residues because treatment of cells with okadaic acid, a potent inhibitor of PP2A, results in various hyperphosphorylated forms of hSecurin which are extremely unstable, due to the action of the Skp1/Cul1/F-box protein complex ubiquitin ligase (27). One of kinases involved in Securin phosphorylation is glycogen synthase kinase 3E (GSK3E). This kinase regulates the abundance of Securin both in nonstressing conditions and after ultraviolet radiation (28, 29). It seems clear that to ensure both sister chromatid separation and cell response to genotoxic stresses, Securin functions are tightly regulated by several mechanisms, including abundance in the cell and phosphorylation status. In this paper, we mutated most of serines, threonines or tyrosines of hSecurin and we found a single mutation that induced chromosomal instability and enhanced cell invasiveness. The relevance of this finding will be discussed. MATERIALS AND METHODS 5 Plasmids, point mutations and sequencing pCDNA3-2HA-hSec, pCDNA3-2HA-hSecD109G and pCDNA3-2HA-hSecS183A/ S184G were previously described (8, 28, 29). pBABE-puro and pCDNA3 were from Addgene and Invitrogen, respectively. hSecT3A, hSecT16A, hSecS28A/S31A, hSecS39A, hSecS42G/T43A, hSecT49A, hSecT60A, hSecT66A/T71A, hSecS74A/T77A, hSecS87G/S89G, hSecT94A/T97A, hSecS102A/S103A, hSecS107A, hSecY111F, hSecS127A, hSecS142G, hSecS165A, hSecS171G and hSecS176A/S181A were constructed using the “Transformer Site-Directed Mutagenesis Kit” from BD Biosciences (30, 31). Sequencing of point mutations was performed on both strands with an automatic sequencer. Primary tumors, cell culture, drugs, transient and stably transfection, and lysis Frozen tumor tissues comprising one colon adenocarcinoma, one lung adenocarcinoma, one classical seminoma and one corticotroph pituitary adenoma were retrieved from cryogenic storage at -80ºC and used following local ethical policies. Informed consent was requested from patients at the time of surgery. Routinely, HCT116 (32) and derivatives, and Cos-7 (from ATCC) cells were grown in Dulbecco’s modified Eagle’s medium (Lonza) as described (8). For some experiments, cells were pretreated with the serine/threonine protein phosphatase inhibitor okadaic acid (1 PM, Sigma) or both okadaic acid and the proteasome and calpain inhibitor Ac- LLnL-CHO (100 PM, Sigma). Where indicated, cells were UV irradiated with 100 J/m2 and harvested 1 hour 30 minutes later (8). Half-life experiments were performed by transient transfection of Cos-7 cells. Expression of the transgene was induced for 18h, cycloheximide (50 Pg/ml) added to the medium and cells harvested at the indicated times. 6 DNA constructs were transiently transfected by electroporation into Cos-7 cells or using the Lipofectamine method into HCT116sec-/- cells (32). At indicated times posttransfection, cells were harvested and lysed. HCT116sec-/- cells were stably transfected with different hSecurin mutants using the puromycin-resistance from pBABE-puro as selection marker. Frozen tissues (tumor cell content over 75%) were disrupted and homogenized in Nonidet P-40 (NP40) lysis buffer (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol and 1% NP40) containing protease inhibitors (1:100 dilution of Protease Inhibitors Cocktail, Sigma). Cell lysis was performed at 4°C using the same buffer. When necessary, extracts were treated with lambda protein phosphatase (18.5 Units/assay). Protein concentration was determined using the Bradford assay. Flow cytometric analysis of cell cycle (FACS) Cells were trypsinized and fixed in 70% ethanol. Propidium iodide staining of nuclei was performed with a CycleTest Plus DNA reagent kit (BD Biosciences), and the DNA content was measured with a FACScan instrument (BD Biosciences). Data were acquired with CellQuest Pro software (BD Biosciences). Small interfering RNA Small interfering RNA (siRNA) transfections were carried out using the Dharmafect reagent (Thermo Fisher Dharmacon) according to the manufacturer’s instructions. Nontargeting (NT) control siRNA and hSecurin specific siRNA were from Thermo Fisher Dharmacon. All siRNAs were used at 100 nM. 7 Electrophoresis, Western blot analysis and antibodies Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and gels were electroblotted onto nitrocellulose membranes and probed with anti-HA (Roche), anti-Securin (Santa Cruz) or anti-D tubulin monoclonal (Sigma) antibodies. Peroxidase-coupled sheep anti-mouse IgG was obtained from GE Healthcare. Immunoreactive bands were visualized using the Enhanced Chemiluminescence Western blotting system (ECL, GE Healthcare). Fluorescence in situ hybridization (FISH) Cultured cells were imprinted onto silanized slides, fixed in ice-cold methanol/glacial acetic acid (3:1) for 10 minutes and air-dried. Then, slides were immersed in a 2x SSC/0.3% NP40 solution at 37ºC during 30 minutes, dehydrated in increasing graded ethanols and air-dried. Cellular DNA and centromeric probes for chromosomes 8 (Spectrum red), 11 (Spectrum green) and 17 (Spectrum acqua) from Vysis were codenatured at 72ºC for 5 minutes and hybridized at 37ºC overnight in a humid chamber (Thermomixer, Eppendorf). After hybridization, slides were washed in 2x SSC/0.3% NP40 solution at 72ºC for 5 minutes, counterstained and mounted with DAPI/antifade (Vysis) and visualized under fluorescence microscopy equipped with appropriate filter sets and a digital camera (Leica). Tumor cell migration and invasion assay The invasion chamber consists of a 24 well plate with control inserts containing an 8 Pm pore-size polyethylene terephthalate (PET) membrane and Matrigel inserts (BD Biosciences) containing an 8 Pm pore-size PET membrane coated with a thin layer of extracellular matrix (ECM). Cells were suspended in serum-free DMEM and added to 8 the upper chamber at 3.5x104 cells/insert. The lower chamber was filled with medium containing 5% fetal bovine serum as chemoattractant. After 48 hours of culture, the upper surface of the inserts was wiped with cotton swabs, and the inserts were stained with Quick Panoptic (QCA) and mounted onto microscopy slides. Cells that migrated through the Matrigel and the PET membrane to the lower surface were photographed under a light microscope and counted with the aid of Image J software (Rasband, W.S., U.S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2011). Percent of invasion was calculated as the number of cells migrating through the Matrigel matrix relative to the migration through the PET membrane. Analysis of gene expression by microarray A human extracellular matrix and adhesion molecules PCR array was purchased from SuperArray Bioscience. The kit determines expression of 84 genes involved in cell-cell and cell-matrix adhesion and housekeeping genes as controls. The array compared gene expression in colon cancer cells stably transfected with pCDNA3-2HA-hSec or pCDNA3-2HA-hSecT60A (clone C12). Briefly, total RNA from each cell line was reverse-transcribed, and the corresponding cDNA was biotin-labeled, as per manufacturer's instructions. The cDNA was denatured, and hybridization was carried out in GEHyb solution to nylon membranes spotted with gene-specific fragments overnight at 60°C. After washing, blots were exposed to x-ray film. GEArray Analyzer version 1.2 was used for analysis. RESULTS Mutation of threonine 60 of Securin augments the protein stability 15 ACKNOWLEGMENTS This work was supported by Spanish grants from Ministerio de Ciencia e Innovación, (SAF2011-30003), Ministerio de Sanidad (Red de Biobancos RD09/0076/00085), Dirección General de Investigación, Tecnología y Empresa, J.A. (P08-CVI-03603, P10- CTS-06243) and Consejería de Salud, J.A. (PI-2009-0589, AI-2011-001). MM-S and CC were supported by fellowships from MICINN; JH-R, SG and MCL-M by J.A. and CS by a contract from ISCIII/FIS and J.A. REFERENCES 1. Hirano T, Funahashi S, Uemura T, Yanagida M. Isolation and characterization of Schizosaccharomyces pombe cutmutants that block nuclear division but not cytokinesis. EMBO J 1986;5(11):2973-9. 2. Yamamoto A, Guacci V, Koshland D. Pds1p is required for faithful execution of anaphase in the yeast, Saccharomyces cerevisiae. J Cell Biol 1996;133(1):85-97. 3. Yamamoto A, Guacci V, Koshland D. Pds1p, an inhibitor of anaphase in budding yeast, plays a critical role in the APC and checkpoint pathway(s). J Cell Biol 1996;133(1):99-110. 4. Zou H, McGarry TJ, Bernal T, Kirschner MW. Identification of a vertebrate sister-chromatid separation inhibitor involved in transformation and tumorigenesis. Science 1999;285(5426):418-22. 16 5. 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Equal amounts of extracts were resolved by SDS-PAGE and immunoblotting performed with anti-HA antibody. C. Cos-7 cells were transiently transfected with the indicated plasmids and expression induced for 18h. Later, cycloheximide (CHX) (50 Pg/ml) was added to the medium and cells were collected at the indicated times (n=3). Extracts were treated with lambda protein phosphatase (18.5 Units/assay) and analyzed by Western blot. Quantification of Securin protein levels was carried out using ImageJ software: 2HA-hSec (dark grey line), 2HA-hSecT60A (light grey line). Error bars represent the standard deviation. Figure 2. Stable expression of 2HA-hSecT60A in HCT116sec-/- cells induces chromosomal instability. A. Study of 2HA-hSec or 2HA-hSecT60A expression levels in stably transfected HCT116sec-/- cells. C6, C11 and C12 are three independent clones stably expressing 2HA-hSecT60A. Extracts were Western blotted for HA and D tubulin detection. B. The cell cycle profile of the three clones was analyzed by FACS analysis. In a parallel manner, cells were subjected to FISH analysis using three centromeric 22 probes as described in Materials and Methods. C. The number of FISH signals per cell was quantified in 100 cells for each determination, and percentage of diploid and tetraploid cells, according to the criteria described in Results, represented in the histogram (D). E. Comparison of FACS analysis of the indicated stably transfected cells, and FISH assays of HCT116sec-/- and 2HA-hSec or 2HA-hSecT60A C12 stably transfected HCT116sec-/- cells using the three centromeric probes described. Figure 3. Transient expression of 2HA-hSecT60A in HCT116sec-/- cells also induces chromosomal instability. HCT116sec-/- cells were transiently transfected with pCDNA3-2HA-hSec or pCDNA3-2HA-hSecT60A and harvested 3 and 7 days later. Expression of transgenes was analyzed by Western blots (A) and their effects on chromosomal instability studied by FISH (100 cells/condition) (B and C). D. The percentage of diploid and tetraploid cells, according to the criteria described in Results, was represented in the histogram. Figure 4. Mutation of threonine 60 of Securin increases cell invasiveness. A. Cell invasion of the indicated cell lines in the presence or absence of extracellular matrix (ECM) was carried out as described in Material and Methods. B. The percent invasion expresses the percent of mean of cells invading through Matrigel insert membrane divided by the mean of cell migrating through control insert membrane. Cells were quantified using ImageJ software. This experiment was repeated a second time with similar results. Figure 5. Knock-down of 2HA-hSecT60A reduces cell invasiveness of HCT116sec-/- 2HA-hSecT60A cells (clones C11 and C12). A. HCT116sec-/-2HA-hSecT60A clones 23 C11 and C12 were interfered with the indicated siRNA(s) and, after 48 hours, extracts were analyzed by Western blotting. B. Cell invasion of transient interfered cells in the presence or absence of extracellular matrix (ECM) was carried out as described in Figure 4A. C. The percent invasion was quantified as in Figure 4B. This experiment was also repeated a second time with similar results. Figure 6. Real-time RT-PCR analysis of MMP7, MMP13, LAMA3, ITGB3, ECM1 and CTGF mRNAs in HCT116sec-/-2HA-hSec (WT) and HCT116sec-/-2HA- hSecT60A clone C12 (C12). Total RNA from each cell line were isolated and the relative mRNA levels of matrix metallopeptidase 7 (MMP7), matrix metallopeptidase 13 (MMP13), laminin alpha 3 (LAMA3), integrin beta 3 (ITGB3), extracellular matrix protein 1 (ECM1) and connective tissue growth factor (CTGF) were determined by realtime RT-PCR. Values are means ±S.D. (n=3). Supplementary Data. Differential expression levels of genes involved in cell-cell and cell-matrix adhesion in HCT116sec-/-2HA-hSecT60A clone C12 vs. HCT116sec-/-2HA- hSec. Figure1 Click here to download high resolution image Supplementary Figure (online publication only) Click here to download high resolution image