Functional Genetic Screens Identify Genes Essential for Tumor Cell Survival… — Volkswagen Polo Vivo

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Functional Genetic Screens Genes Essential for Tumor Survival in Head and Neck and Cancer

Abstract

Purpose: continuous improvement of treatment the mortality rates for non–small lung cancer (NSCLC) and and neck squamous cell (HNSCC) remain disappointingly and novel anticancer agents are awaited.

Experimental Design: We the data from genome-wide screens on tumor cell in a lung and a head and neck cell line.

Results: We 71 target genes that essential for the survival of both types. We identified a cluster of 20 that play an important during G 2 –M phase transition, the importance of this cell-cycle for tumor cell survival. genes from this ( CKAP5 . KPNB1 . RAN . TPX2 . and ) were evaluated in more and have been shown to be for tumor cell survival in tumor types, but most in HNSCC. Phenotypes that observed following siRNA-mediated of KIF11 (kinesin family 11) were reproduced by inhibition of using the small-molecule inhibitor (SB-715992). We showed that induces a G 2 arrest, causes chromosome segregation, and induces death in HNSCC in vitro . primary keratinocytes are less Furthermore, growth of HNSCC engrafted in immunodeficient mice was inhibited after ispinesib

Conclusion: This study a wide array of druggable for both lung and head and cancer. In particular, multiple involved in the G 2 –M checkpoint were to be essential for tumor cell indicating their potential as targets. Clin Cancer 19(8); 1994–2003. ©2013 .

Translational Relevance

The prognosis of and head and neck cancer is disappointing and novel treatments are awaited. In this study, we a genome-wide siRNA screen to genes that seem to be for tumor cell viability. A subgroup of these genes was to G 2 –M regulation of the cell cycle and was for their suitability as targets to lung and head and neck cells. A drug against one of genes, KIF11 . was tested in mouse models and inhibited growth significantly. In summary, we that genome-wide siRNA deliver a multitude of druggable that can be exploited to improve of both lung and head and cancer.

Introduction

Two of the more diagnosed types of cancer in the are those in the lung and the head and region. Lung cancer is the most common cancer in the whereas head and neck is the sixth most common worldwide (1, 2 ). The predominant histologic of head and neck cancer is cell carcinoma (HNSCC), is found in more than 95% of the (3 ). Lung cancer is more as multiple different histologic of tumor cells can be distinguished. cancers are classified as non–small lung cancer (NSCLC) and cell lung cancer which make up approximately 80% and 20% of the number of cases, respectively (4 ). can be further subdivided into histologic subtypes, of which cell carcinomas (40%) and (37%) are the most predominant (4 ).

Advanced stages of lung and and neck cancer are often by a combination of platinum-containing chemotherapy and radiotherapy. Despite improvement of control, the current survival of patients with both and head and neck cancer disappointing. For HNSCC, the 5-year rate is approximately 50% to 60% and this has increased slightly during the 3 decades (5 ). For lung cancer, the is even worse with a survival rate of only 5% to 15% (6 ). there is an urgent need to current therapies. The recent with targeted drugs that identification of druggable that are essential for tumor may fuel the development of novel approaches (7–9 ).

To survive and tumor cells strongly on specific genetic and epigenetic These tumorigenic alterations are for important cancer-associated phenotypes, as deregulation of apoptosis and cell-cycle (10 ). Because these genetic drive tumorigenesis, they become the Achilles’ heel of the Moreover, the rewiring of signaling by alterations in the participating genes may that expression of some becomes very critical for cell survival. Several have already reported tumor cell survival by of individual genes in a specific of somatic mutations, a phenomenon referred to as synthetic lethality ). Therefore, the identification of genes for cell viability and the unmasking of lethal interactions in tumor provide a powerful approach for the of novel therapeutic targets.

RNA interference (RNAi) screens are suited for the discovery of genes for tumor cell survival. in this study, we explored siRNA screen data and a total of 362 tumor lethal Strikingly, many of the potential essential genes targeted by siRNAs are involved in the regulation of the G 2 –M of the cell cycle. Several of candidates were validated as anticancer targets.

Materials and

Cell lines and animal

The cancer cell lines and fibroblasts were cultured in Modified Eagle’s Medium 5% fetal calf serum and 2 mmol/L l -glutamine (Lonza). keratinocytes were cultured in serum-free medium (Invitrogen) with 0.1% bovine albumin (BSA), 25 mg bovine extract, 2.5 μg human recombinant 250 μg Amphotericin B (MP biomedicals), and 250 μg gentamycin Cells were grown in a atmosphere of 5% CO 2 at 37°C.

NSCLC lines SW1573, A549, and H1299 were obtained the American Type Culture HNSCC cell lines UM-SCC-22A, and UM-SCC-22B were from Dr. T Carey (University of Ann Arbor, MI; ref. 14 ). Cell VU-SCC-120 (formerly known as and VU-SCC-OE were established as previously (15 ). The HNSCC cell were authenticated to the earliest passages by microsatellite profiling and mutation analysis (14. 16 ). keratinocytes and human fibroblasts isolated from an uvulopalatopharyngoplasty and served as normal control. Use of tissue from surgical was according to the guidelines of the Dutch Scientific Societies (www.federa.org ) and the law on medical research. Informed was obtained of enrolled patients, required.

siRNA screens

The SW1573 cell line was subjected to a forward transfection in 96-well (Cellstar, Greiner Bio-One). were seeded using a microplate dispenser (Bio-Tek) and 24 later, the cells were on an automated platform. In total 25 of each siRNA SMARTpool from the siARRAY Human library [Catalog items (Sept05), G-003600 (Sept05), (Sept05) and G-005000 (Oct05); Thermo Fisher Scientific) and μL DharmaFECT1 (Thermo Fisher were delivered to the cells the Sciclone ALH 3000 workstation LifeSciences) and a Twister II microplate (Caliper LifeSciences). The nontargeting and the PLK1 SMARTpool were as negative and positive control, Plates were incubated for 96 at 37°C/5% CO 2 . Afterwards, the cells fixed and stained for 1 hour a 3.7% formaldehyde solution in H 2 O 0.5 μg/mL Hoechst 33342. The of cells was determined using the eX3 microplate cytometer (TTP by automatically counting the number of present in each well.

cells were plated and in 96-well flat-bottom low evaporation TPP (VWR International) using the automated platform and assay Cells were transfected 25 nmol siRNA and 0.03 μL Cell viability was determined by CellTiter-Blue Reagent (Promega) a Multidrop Combi (Thermo Scientific) in cell culture After 2 hours of incubation at fluorescence was analyzed at 540 nm excitation and 590 nm wavelength using an Infinite microplate reader (Tecan).

of cancer-lethal siRNA pools

To the potency of the obtained hits, 3 cell lines (A549, and H460) and 3 HNSCC cell (UM-SCC-11B, UM-SCC-22B, and VU-SCC-120) transfected with siRNAs a variety of G 2 –M phase-related genes. The siCONTROL#2 and the PLK1 SMARTpool used as negative and positive respectively. All cell cultures transfected with 25 nmol and DharmaFECT1. NSCLC cell SW1573 was transfected with μL DharmaFECT1, cell lines and H1299 with 0.03 μL and μL, respectively. HNSCC cell UM-SCC-11B was transfected with μL DharmaFECT1, UM-SCC-22B and VU-SCC-120 0.15 μL and 0.03 μL, respectively. viability was measured 96 hours transfection using CellTiter-Blue (Promega) as described above.

RT-PCR

Western blot

Cell-cycle analysis

Cells treated with 4 nmol/L (Selleck Chemicals) during 24 after which the cells incubated with 4 nmol/L (BrdUrd; Sigma-Aldrich) for 45 minutes. were subsequently harvested and overnight in 70% EtOH. Next, were incubated with 0.5 RNAse A in PBS at 37°C. After 30 cells were washed and in 5 mol/L HCl with 0.5% X-100. The cells were for 20 minutes at room temperature, which the solution was neutralized by of 0.1 mol/L Na 2 B 4 O 7 . The cells were for BrdUrd incorporation using anti-BrdUrd antibodies, followed by isothiocyanate (FITC)-conjugated rabbit antibodies (Dako) in PBS with Tween-20 and 1% BSA. Staining for DNA was conducted using propidium The cell-cycle distribution was analyzed a BD FACSCalibur flow cytometer (BD Cell-cycle analyses were using BD CellQuest software (BD

Staining of mitotic spindles

were grown on an 8-wells Chamber Slide (Thermo Scientific) and treated with 4 ispinesib (Selleck Chemicals) for 24 The cells were fixed 1 hour in 4% formaldehyde (Fluka and subsequently permeabilized with Triton-X100 (ICN Biochemicals). An antibody (clone B-7; Cruz Biotechnology) was applied in a dilution for 40 minutes to visualize spindles. A FITC-labeled anti-mouse (Dako) was used as secondary The DNA was visualized with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). The slides mounted with fluorescence medium (Dako).

Efficacy of in vivo

All animal experiments carried out according to the NIH Principles of Animal Care and Dutch law (Wet op de dierproeven, Stb 1985,

Results

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A large panel of is involved in cell viability

The cell line SW1573 and the cell line VU-SCC-120 subjected to an optimization procedure for high-throughput forward siRNA An siRNA SMARTpool targeting . a gene essential for cancer viability (18, 19 ), was used as control. Optimal transfection resulted in a reduction of at least 70% viability in the PLK1 siRNA-transfected as compared with siCONTROL#2, a siRNA. In addition, more 80% gene-specific knockdown was observed by real-time PCR (qRT-PCR; data not Introduction of siCONTROL#2 did not reduce viability with more 10% to 20% compared with untransfected indicating that the transfection did not lead to excessive nonspecific death.

Identification of genes for NSCLC and HNSCC tumor viability by genome-wide siRNA A, Z -score calculations were using cell counts top) or cell viability (HNSCC, bottom). Black represent the Z -score for individual pools that target one PLK1 siRNAs were as positive controls and are indicated as dots (encircled). Nontargeting (siCONTROL) were used as controls and are shown in gray. The Z = threshold (black line) was to determine siRNAs that influenced tumor cell ( P 0.003). Z -scores were using the mean value per from the 2 independent siRNA B, a cluster of 20 genes involved in the spindle checkpoint was identified the 362 siRNAs that decreased viability in NSCLC and/or

To identify common pathways are essential for tumor cell we subjected the obtained hits to analysis. First, we clustered the 71 that were found to be for tumor cell survival in NSCLC and HNSCC using the database (version 9.0), and revealed 3 clusters that genes involved in RNA processing, biogenesis, and protein modification/ubiquitination Fig. S1A). Because we a stringent cutoff of Z = −2.75, it is that we excluded siRNAs do give a lethal phenotype in one of the cell lines, whereas it did not reach the cutoff in the other line. Therefore, we also the NSCLC and the HNSCC hit lists for analysis. This analysis 362 hits in total, which contained the same clusters, but many more genes per (Supplementary Fig. S1B). One consisted of 20 genes involved in the of the G 2 –M phase of the cell cycle 1B ). It is known that in both and HNSCC, the cell-cycle checkpoints at G 1 and G 2 are inactivated by abrogation of the p53 and pRb pathways. The identified in the G 2 –M phase might relate to these specific and we therefore analyzed these in detail.


Mitotic spindle assembly and is vital

Figure 2.

Deconvolution of pools that decreased and HNSCC cell viability. A, NSCLC (A549, H1299, and and 3 HNSCC (VU-SCC-120, UM-SCC-11B, and cell lines were with the KIF11 siRNA and the 4 individual siRNAs to determine the of the lethal phenotype observed in the screens. Cell viability was in triplicate and calculated relative to cells. Bars are median viability values and error represent the SD. All HNSCC cell showed the lethal phenotype at least 3 out of 4 siRNAs. All NSCLC lines showed similar except for SW1573. B, the AURKA and individual siRNAs were but showed a less convincing killing phenotype than siRNAs.

One of the strongest and most hits was RAN . However, 3 genes with spindle formation via RAN ( DLG7 . NUTF2 . and RCC1 ) not identified as putative hits in the screens. In subsequent deconvolution siRNAs targeting these indeed confirmed that role does not seem to be for cell survival (Supplementary S2F–S2H).

Surprisingly, aurora did not seem to be lethal tumor in the genome-wide screens, even AURKA . AURKB . and AURKC emerged as key mitotic regulators ) and have been explored as drug targets (23 ). We therefore the siRNA pools of the AURK and analyzed the cell viability in 3 and 3 NSCLC cell lines 2B and Supplementary Fig. S4A and S4B). inhibition showed an effect cell death) on the cell of 3 of 6 cell lines tested, AURKB and AURKC did not show a in any of the cell lines. We tested the of knockdown induced by all siRNAs and concluded that the lack of is not technical as all siRNAs showed mRNA knockdown (Supplementary S4C and S4D). Next, we explored the that the aurora kinases a redundant function by simultaneous of these genes (Supplementary S4E and S4F). None of the combinations in an increase in cell death. many hits identified in the screens could be validated, more or less expected that were not found also not confirmed in deconvolution This observation, although on limited numbers of genes, the accuracy of the obtained hit list of tumor lethal siRNAs.

Functional KIF11 is essential for viability

We showed that genes that act during the G 2 –M of the cell cycle are apparently for the viability of NSCLC and HNSCC which makes these potential targets for therapy 2A ). These genes included the motor protein KIF11 . of cell lines with (SB-715992), a potent and highly small-molecule inhibitor of KIF11 (24 ), that functional KIF11 is for cell viability (Fig. 3 ). treated with ispinesib, human oral keratinocytes and only showed a mild inhibition (approximately 40% compared untreated controls), whereas lines derived from HNSCCs were completely in their growth (Fig. 3B ). NSCLC cell lines growth inhibition when was applied (Fig. 3A ), but this seemed less dramatic compared with the HNSCC lines. Cell line was only marginally affected in its after ispinesib incubation, is in line with the observation knockdown of KIF11 expression in only a minor growth effect in this cell

Figure 3.

KIF11 is important for and particularly HNSCC cell A, four NSCLC cell (black lines) were with 18 concentrations of ispinesib a small-molecule inhibitor of KIF11. All lines showed growth after ispinesib treatment, primary oral keratinocytes and (gray lines) only mild effects. SW1573 was the insensitive cell line the NSCLC panel. B, five cell lines were to 18 concentrations of ispinesib. All 5 cell (black lines) showed growth inhibition. C, the KIF11 level of cultured HNSCC and cell lines was determined. keratinocytes (represented by the black showed very low KIF11 as compared with the tumor lines. The NSCLC cell (A549, H1299, SW1573, and showed higher KIF11 levels than the HNSCC lines (VU-SCC-120, UM-SCC-11B, and This corresponds to the lower to ispinesib seen in A and B. Bars median values of 3 independent and error bars represent the SD.

we checked the basal expression of KIF11 in all NSCLC and HNSCC lines used and determined the overall KIF11 expression was higher in the NSCLC cell ( P = 4.44 × 10 −6 ; Fig. 3C and Supplementary S5) than in the HNSCC cell This suggests that the phenotype after KIF11 or drug inhibition in NSCLC lines might be the result of KIF11 expression. On the other the expression of KIF11 in SW1573 not differ from the other cell lines, suggesting the lack of response to ispinesib is not related to an exceptionally high expression in SW1573.

On the basis of the tumor cell phenotype after siRNA as well as ispinesib treatment, we that particularly HNSCC lines are tremendously sensitive to inhibition and in subsequent experiments we on these cell lines. inhibits the interaction between and microtubules, thereby blocking the of a functional bipolar mitotic leading to cell-cycle arrest in and subsequent cell death (24 ). We the improper mitotic spindle in the presence of ispinesib in 2 HNSCC lines. Indeed, 100 of 100 dividing cells (100%) treated ispinesib showed monopolar spindles (Fig. 4A ), whereas 55 of 202 primary keratinocytes (27%) monopolar spindles ( P 0.001, exact probability test). In we examined the cell-cycle distribution in cell lines in the presence of and found accumulation of cells in G 2 (Fig. 4B and Supplementary Fig. As expected from the growth experiments with ispinesib, did not show G 2 arrest to the same This indicates that seems a suitable HNSCC target with less on nontumorigenic cells.

Inhibition of impairs mitotic spindle and induces G 2 arrest. A, HNSCC line VU-SCC-OE showed mitotic spindles (top), the bipolar mitotic spindles replaced by monopolar spindles incubation with ispinesib at second row). Also, the of the condensed chromosomes at the metaphase is abrogated after ispinesib Pictures were taken a ×40 magnification. Primary keratinocytes normal mitotic spindles row) and this was hardly after ispinesib treatment B, HNSCC cells treated ispinesib showed accumulation of in the G 2 –M phase of the cell cycle as with untreated cells. In fibroblasts, this increase in G 2 was significantly less compared the tumor cell lines ( P for both cancer cell Fisher exact probability Measurements were carried out in 2 experiments.

Efficacy of ispinesib in HNSCC cancer models

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