An RNA interference screen for identifying downstream effectors of the p53 and pRB tumour suppressor pathways involved in senescence

  • Emilie Rovillain1,

    Affiliated with

    • Louise Mansfield1,

      Affiliated with

      • Christopher J Lord2,

        Affiliated with

        • Alan Ashworth2 and

          Affiliated with

          • Parmjit S Jat1Email author

            Affiliated with

            BMC Genomics201112:355

            DOI: 10.1186/1471-2164-12-355

            Received: 14 December 2010

            Accepted: 8 July 2011

            Published: 8 July 2011

            Abstract

            Background

            Cellular senescence is an irreversible cell cycle arrest that normal cells undergo in response to progressive shortening of telomeres, changes in telomeric structure, oncogene activation or oxidative stress and acts as an important tumour suppressor mechanism.

            Results

            To identify the downstream effectors of the p53-p21 and p16-pRB tumour suppressor pathways crucial for mediating entry into senescence, we have carried out a loss-of-function RNA interference screen in conditionally immortalised human fibroblasts that can be induced to rapidly undergo senescence, whereas in primary cultures senescence is stochastic and occurs asynchronously. These cells are immortal but undergo a rapid irreversible arrest upon activation of the p53-p21 and p16-pRB pathways that can be readily bypassed upon their inactivation. The primary screen identified 112 known genes including p53 and another 29 shRNAmirs targetting as yet unidentified loci. Comparison of these known targets with genes known to be up-regulated upon senescence in these cells, by micro-array expression profiling, identified 4 common genes TMEM9B, ATXN10, LAYN and LTBP2/3. Direct silencing of these common genes, using lentiviral shRNAmirs, bypassed senescence in the conditionally immortalised cells.

            Conclusion

            The senescence bypass screen identified TMEM9B, ATXN10, LAYN and LTBP2/3 as novel downstream effectors of the p53-p21 and p16-pRB tumour suppressor pathways. Although none of them has previously been linked to cellular senescence, TMEM9B has been suggested to be an upstream activator of NF-κB signalling which has been found to have a causal role in promoting senescence. Future studies will focus on determining on how many of the other primary hits also have a casual role in senescence and what is the mechanism of action.

            Cellular senescence RNA interference screen senescence bypass conditionally immortal cells

            Background

            Normal somatic cells undergo a finite number of divisions before entering a state of irreversible growth arrest termed cellular senescence [1]. This is triggered in response to a variety of intrinsic and extrinsic stimuli including progressive telomere shortening or changes in telomeric structure at the ends of chromosomes or other forms of genotoxic stress such as oncogene activation, or DNA damage or oxidative stress, resulting in a DNA damage response and growth arrest via activation of the p53 tumour suppressor pathway [2, 3]. Non-genotoxic stress induces senescence by a telomere independent mechanism involving activation of the p16-pRB pathway by up-regulation of p16INK4a [3, 4].

            Cellular senescence acts as an important tumour suppressor mechanism. Overcoming senescence and acquiring a limitless replicative potential has been proposed to be one of the key events required for malignant transformation [5]. Senescence is thought to have evolved as an example of antagonistic pleiotrophy, whereby its beneficial traits in a reproductively active individual have deleterious effects later in life [6, 7]. The underlying mechanisms that control cellular senescence, the signal transduction pathways involved and how the diverse signals that result in senescence are all integrated, remain poorly defined. Moreover the downstream effectors of the p53-p21 and p16-pRB pathways that result in the irreversible growth arrest and entry into senescence are unknown.

            The discovery of RNA interference as a mechanism to silence gene expression has revolutionized studies on mammalian gene expression and has permitted loss-of-function genome-wide genetic screens, to identify genes involved in a variety of cellular processes, to be performed [812]. A number of shRNA libraries have been developed for carrying out such genome-wide screens, one of which is the pSM2 Retroviral shRNAmir library [13] (Thermo Scientific Open Biosystems). This library has several unique features that make it very versatile and efficient for large-scale screens particularly the human microRNA-30 (miR30) adapted design which increases knockdown specificity and efficiency [14].

            Here we present a RNA interference screen using the human pSM2 retroviral shRNAmir library, carried out in the conditionally immortal HMF3A human fibroblasts, to identify genes whose silencing bypasses senescence arrest induced by activation of the p53-p21 and p16-pRB pathways. The primary screen identified 112 known genes and another 29 shRNAmirs targetting as yet unidentified loci. Comparison of the known targets with genes known to be up-regulated upon senescence by micro-array expression profiling, identified 4 common genes whose expression was reversed when senescence was bypassed upon inactivation of the p53-p21 and p16-pRB pathways.

            Results

            To directly identify the downstream effectors of the p53-p21 and p16-pRB pathways, we have utilized the conditionally immortal HMF3A human fibroblasts that were derived by immortalising adult human mammary fibroblasts with the catalytic subunit of human telomerase and a thermolabile U19tsA58 mutant of SV40 Large T antigen [15]. These cells are immortal if grown at 34°C but undergo a senescence arrest upon inactivation of the thermolabile U19tsA58 T antigen resulting in the activation of the p53-p21 and p16-pRB pathways [15]. They are stringently temperature sensitive but senescence can be readily bypassed by inactivation of the p53-p21 or the p16-pRB pathway [16]. To facilitate efficient transduction of these cells by retroviral infection, they were transduced with the full length murine ecotropic retroviral receptor and CL3EcoR cells derived, that most closely mirror the parental cells [16]. The temperature dependent senescent arrest of CL3EcoR cells and its bypass upon inactivation of p21CIP1 by silencing or sequestration of the RB family of proteins by HPV16 E7 are shown in Figure 1.
            http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-355/MediaObjects/12864_2010_3526_Fig1_HTML.jpg
            Figure 1

            Characteristics of CL3 EcoR cells. a: CL3EcoR cells are immortal at 34°C but undergo a senescence arrest upon shift up 38°C. b: Senescence is bypassed upon silencing of p21CIP1 using pRSp21F or sequestration of RB family of proteins by HPV16 E7.

            The pSM2 library version 1.3 comprising 15,148 constructs targetting 9,392 human cancer associated genes was amplified and each 96 well plate used to prepare a pool of plasmid DNA; each of the 100 pools contained between 150 to 200 different shRNAmir constructs with each gene being represented by 1 to 3 shRNAmirs. To ensure that CL3EcoR cells were sufficiently sensitive to identify a single shRNAmir construct in a pool of 200 shRNAmir constructs, pRSp21F (a p21CIP1 shRNA construct) [17] was mixed in a ratio of 1:200 with pRSLaminA/C and used to assay bypass of senescence in CL3EcoR cells. The pRSp21F construct was used because the pSM2 library version 1.3 did not contain any silencing constructs for p21CIP1. Silencing of LaminA/C did not bypass senescence, very few growing colonies were obtained (Figure 2a) whereas silencing of p21CIP1 was very efficient and produced essentially a confluent monolayer of growing cells (Figure 2b). The 1:200 p21CIP1/LaminA/C mix produced numerous distinct densely growing colonies (Figure 2c) indicating that CL3EcoR cells and the procedure were sufficiently sensitive to generate colonies in which senescence had been overcome.
            http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-355/MediaObjects/12864_2010_3526_Fig2_HTML.jpg
            Figure 2

            Sensitivity of the screen. CL3EcoR cells were infected with retroviruses prepared from pRSLamin A/C (a), pRSp21F (b), or a 1/200 mix of pRSp21F/pRSLamin A/C (c). After puromycin selection, cells were reseeded at 8.5 × 104 per 15 cm plate or 0.5 × 104 per well in 6-well plates and shifted to 38°C for 3 weeks.

            ShRNA interference screen

            The formula: ln [1-0.95]/ln [1-1/(Library Size)] recommended for genetic screens by the Nolan lab (http://​www.​stanford.​edu/​group/​nolan/​screens/​screens.​html), suggested that approximately 1000 independent infectious events would be sufficient for a 99% confidence that all shRNAs within a pool had been sampled. To ensure that the screen would be saturating, virus sufficient to yield 10,000 infectious events was utilised for each pool (shown in Additional File 1). The screen was performed in successive waves of 10 pools. To minimise variation and background reversion, CL3EcoR cells were used at the same passage for every pool. Virus prepared from pRSp21F and pRSLamin A/C was used as positive and negative controls respectively to evaluate the level of background and ensure that the complementation assay worked for each round of the screen. Stably transduced cells were trypsinised and reseeded. Three weeks after reseeding, flasks were examined to identify growing colonies; a representative colony is shown in Figure 3. Each colony was examined microscopically to ensure it comprised growing cells and the number of colonies obtained for each pool determined. The number of stably transduced cells, the number of flasks reseeded and the number of colonies obtained for each flask at 38°C are shown in Additional File 2. The flasks which contained more densely growing/bigger colonies (indicated in red in Additional File 2) were trypsinised, replated and used for extracting genomic DNA when confluent.
            http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-355/MediaObjects/12864_2010_3526_Fig3_HTML.jpg
            Figure 3

            ShRNA screen. CL3EcoR cells were transduced with ecotropic retroviruses prepared from each of the 100 pools of shRNAmirs, selected with puromycin, reseeded and shifted up to 38°C for 3 weeks. Densely growing colonies were considered to have bypassed senescence and potentially contain candidate shRNAmirs.

            34 out of 100 pools yielded healthy growing colonies; pools 13, 78 and 82 particularly gave a higher number of colonies which were also larger. Pools 16, 18, 19, 21 and 80 also yielded colonies but they were smaller. For each pool that contained growing colonies, 1 to 4 flasks containing the highest number of growing colonies, were reseeded for extracting genomic DNA and resulted in a total of 81 sub-pools.

            Identification of shRNAmirs

            The shRNAmir proviral inserts were by amplified by two rounds of nested PCR using pSM2 specific primers, the amplified products TOPO cloned and plasmid DNA extracted from at least six colonies sequenced to identify all shRNAs present within each pool; for some pools, DNA from many more colonies was sequenced. The hair pin sequence was determined by searching for the miR-30 context and the miR-30 loop that are common to all inserts and frame the hair pin. The sequence of the hair pin was used to identify the target gene by searching the pSM2 data base or by BLASTN analysis of the NCBI human genome data base (http://​blast.​ncbi.​nlm.​nih.​gov/​Blast.​cgi). Sequences that could not be linked back to the list of hair pin sequences within the Open Biosystems collection or were not 100% homologous to a gene within Genbank were not pursued and are presented in Additional File 3.

            The rescued shRNAmir hair pins identified 112 known genes and another 29 shRNAmirs targetting as yet unidentified loci. For each pool, the number of times that sequence was obtained, the corresponding insert reference, gene name and the number of shRNAmir constructs for that gene within the SM2 library are shown in Table 1. The last column of the table indicates if the recovered insert was a match to a hair pin present in that particular pool ("match") or if it was present within another pool ("listed in pool X"). 14 hair pins were isolated from incorrect pools. Some inserts were detected several times in multiple pools. For example, the hair pin v2HS_119967 (corresponding to LOC155004) listed within pool 52 was also identified from pool 3, 4, 5, 9, 12, 30, 59, 60, 64, 71, 72, 74, 77, and 98 suggesting that there may have been some cross-contamination of the library perhaps during replica plating. However, this was not a problem since the aim of our screen was to identify shRNAmirs that are able to bypass senescence and does not depend upon the identity of the pool. Since this insert was isolated from so many pools, it could be that it is a strong positive or that it was highly represented within the library and likely to be a false positive.
            Table 1

            Primary screen

            Pool

            Insert

            Gene name

            Gene symbol

            Freq

            Cons per Gene

            Library location

            3

            v2HS_63142

            keratin associated protein 5-9

            KRTAP5-9

            3/9

            1

            match

             

            v2HS_119967

            LOC155004

            LOC155004

            4/9

            2

            pool 52

             

            v2HS_56766

            acyl-CoA synthetase medium-chain family member 3

            ACSM3

            1/9

            2

            match

             

            v2HS_59294

            glycoprotein hormone alpha 2

            GPHA2

            1/9

            1

            match

             

            v2HS_53974

            PRO0255 protein

            PRO0255

            5/8

            1

            match

             

            v2HS_63482

            paired box gene 1

            PAX1

            2/8

            1

            match

            4

            v2HS_119967

            LOC155004

            LOC155004

            2/10

            2

            pool 52

             

            v2HS_66751

            LOC344082

            LOC344082

            4/10

            1

            match

             

            v2HS_70011

            serum amyloid A-like 1

            SAAL1

            2/10

            2

            match

             

            v2HS_98079

            human solute carrier family 22

            SLC22A3

            1/10

            2

            pool 79

            5

            v2HS_108647

            LOC349868

            LOC349868

            3/8

            1

            pool 79

             

            v2HS_70473

            polymerase (DNA directed), mu

            POLM

            2/8

              
             

            v2HS_71958

            human olfactory receptor, family 5, subfamily P, member 3

            OR5P3

            3/8

            1

            match

             

            v2HS_119967

            LOC155004

            LOC155004

            7/12

            2

            pool 52

             

            v2HS_66652

            protein phosphatase 3, catalytic subunit, β isoform

            PPP3CB

            1/12

            1

            match

             

            v2HS_70473

            polymerase (DNA directed), mu

            POLM

            4/12

              

            7

            v2HS_112910

            human cyclin-dependent kinase 8

            CDK8

            1/10

            3

            match

             

            v2HS_98079

            solute carrier family 22, member3

            SLC22A3

            1/10

            2

            pool 79

             

            v2HS_97017

            sterile alpha motif containing 4a

            SAMD4A

            1/10

              
             

            v2HS_69776

            cytochrome P450, family 4, subfamily Z, polypeptide

            CYP4Z2P

            1/10

            2

            match

              

            2 pseudogene

                

            9

            v2HS_108647

            LOC349868

            LOC349868

            1/8

            1

            pool 79

             

            v2HS_119967

            LOC155004

            LOC155004

            1/8

            2

            pool 52

             

            v2HS_62506

            family with sequence similarity 181, member B

            FAM181B

            1/8

            1

            match

             

            v2HS_55950

            prostate stem cell antigen

            PSCA

            2/13

            1

            match

             

            v2HS_119967

            LOC155004

            LOC155004

            3/13

            2

            pool 52

             

            v2HS_70312

            TRK-fused gene

            TFG

            4/13

            3

            match

             

            v2HS_71740

            ataxin 10

            ATXN10

            1/13

            1

            match

             

            v2HS_98079

            solute carrier family 22, member 3

            SLC22A3

            1/13

            2

            pool 79

             

            v2HS_65121

            sorting nexin 12

            SNX12

            1/13

            1

            match

             

            v2HS_64384

            amyloid beta precursor protein binding family A member 2

            APBA2

            1/6

            1

            match

             

            v2HS_97017

            sterile alpha motif containing 4A

            SAMD4A

            1/6

              

            12

            v2HS_119967

            LOC155004

            LOC155004

            5/11

            2

            pool 52

             

            v2HS_58950

            signal-regulatory protein beta 2

            SIRPB2

            3/11

            2

            match

              

            protein tyrosine phosphatase, non-receptor type 13

            PTPN13

            1/10

              
             

            v2HS_119967

            LOC155004

            LOC155004

            5/10

            2

            pool 52

            13

            v2HS_55731

            phenylalanine-tRNA synthetase-like, beta subunit

            FARSLB

            1/12

            2

            match

             

            v2HS_59258

            dynein, light chain, roadblock-type 1

            DYNLRB1

            3/12

            1

            match

             

            v2HS_59891

            TAO kinase 1

            TAOK1

            5/12

            3

            match

             

            v2HS_162164

            iodotyrosine deiodinase

            IYD

            1/12

              
              

            cell cycle exit and neuronal differentiation 1

            CEND1

            1/12

              
              

            transmembrane protein 168

            TMEM168

            2/5

              
             

            v2HS_68714

            abl-interactor 1

            ABL1

            3/5

            3

            match

              

            cell cycle exit and neuronal differentiation 1

            CEND1

            4/6

              
             

            v2HS_58952

            signal regulatory protein gamma

            SIRPG

            1/6

            2

            pool 14

             

            v2HS_59653

            LOC342404

            LOC342404

            1/6

            1

            match

             

            v2HS_55731

            phenylalanyl-tRNA synthetase like, beta subunit

            FARSLB

            1/15

            2

            match

             

            v2HS_64320

            Smith-Magenis syndrome chromosome region, candidate 7

            SMCR7

            1/15

            1

            match

             

            v2HS_71174

            peroxisome proliferator-activated receptor, gamma

            PPARGC1A

            3/15

            1

            match

             

            v2HS_71453

            similar to IAP-associated factor VIAF1; phosducin-like

             

            2/15

            1

            match

            16

            v2HS_68478

            CD28 antigen

            CD28

            8/12

            2

            pool 13

             

            v2HS_63107

            dopamine beta hydoxylase

            DBH

            2/12

            1

            match

             

            v2HS_61750

            LOC100129230

            LOC100129230

            7/13

            3

            match

              

            LOC9142

            LOC9142

            2/13

              
             

            V2HS_62831

            membrane bound O-acyltransferase domain containing 1

            MBOAT1

            3/13

            2

            match

            18

            v2HS_63989

            STAR-related lipid transfer (START) domain containing 6

            STARD6

            1/14

            2

            match

              

            LOC730256

            LOC730256

            1/14

              

            19

            v2HS_58958

            TMEM9 domain family, member B/C11orf15

            TMEM9B

            3/10

            1

            match

             

            v2HS_57051

            RNA binding motif, single stranded interacting protein 1

            RBMS1

            5/10

            1

            match

             

            v2HS_106158

            LOC345672

            LOC345672

            1/10

            2

            pool 82

             

            v2HS_59560

            chromosome 13 open reading frame 15

            C13orf15

            10/10

            1

            match

            21

            v2HS_55312

            glucosamine-phosphate N-acetyltransferase 1

            GNPNAT1

            6/9

            2

            match

             

            v2HS_68437

            LOC157503

            LOC157503

            2/9

            3

            match

            30

            v2HS_119967

            LOC155004

            LOC155004

            6/14

            2

            pool 52

             

            v2HS_34338

            protein phosphatase 4, regulatory subunit 2

            PPP4R2

            7/14

            3

            match

             

            v2HS_119967

            LOC155004

            LOC155004

            1/13

            2

            pool 52

             

            v2HS_36467

            solute carrier family 33 (acetyl-CoA transporter), member 1

            SLC33A1

            8/13

            3

            match

             

            v2HS_46793

            ubiquitin-like modifier activating enzyme 3

            UBA3

            1/13

            1

            match

            32

            v2HS_42104

            citrate lyase beta like (CLYBL), transcript variant 1

            CLYBL

            5/7

            1

            match

              

            LOC100129563

            LOC 10012956

            1/7

              

            41

            v2HS_48278

            choline kinase-like

            CHKL

            6/6

            1

            match

            54

            v2HS_112629

            basic transcription factor 3, like 1

            BTF3L1

            1/5

            1

            match

             

            v2HS_121153

            LOC221399

            LOC221399

            2/5

            1

            match

             

            v2HS_125538

            LOC350103

            LOC350103

            1/5

            1

            match

             

            v2HS_129527

            LOC351851

            LOC351851

            6/6

            1

            match

             

            v2HS_112629

            basic transcription factor 3, like 1

            BTF3L1

            2/6

            1

            match

            55

            v2HS_117465

            SH3 domain binding glutamic acid-rich protein like 2

            SH3BGRL2

            2/6

            1

            match

             

            v2HS_119051

            LOC90841

            LOC90841

            1/6

            1

            match

             

            v2HS_116174

            YTH domain containing 2

            YTHDC2

            1/7

            2

            pool 58

             

            v2HS_119120

            hypothetical protein FLJ20032

            FLJ20032

            6/7

            2

            match

            56

            V2HS_120429

            six-twelve leukemia (STL), non-coding RNA

            STL

            5/6

            1

            match

             

            v2HS_115231

            rab23, member RAS oncogene family

            RAB23

            4/6

            4

            match

             

            v2HS_117914

            transketolase-like 2

            TKTL2

            2/6

            1

            match

             

            v2HS_117239

            chromosome 9 open reading frame 58

            C9orf58

            3/6

            1

            match

             

            v2HS_120429

            six-twelve leukemia (STL), non-coding RNA

            STL

            1/6

            1

            match

             

            v2HS_119206

            lon peptidase 2, peroxisomal

            LONP2

            2/6

            1

            pool 146

            58

            v2HS_116174

            YTH domain containing 2

            YTHDC2

            1/4

            2

            match

             

            v2HS_118722

            layilin

            LAYN

            2/4

            2

            match

             

            v2HS_112838

            ectonucleoside triphosphate diphosphohydrolase 3

            ENTPD3

            3/7

            4

            match

             

            v2HS_112982

            chromodomain helicase DNA binding protein 3

            CHD3

            1/7

            3

            match

             

            v2HS_116174

            YTH domain containing 2

            YTHDC2

            1/7

            2

            match

             

            v2HS_112838

            ectonucleoside triphosphate diphosphohydrolase 3

            ENTPD3

            1/12

            4

            match

             

            v2HS_118254

            WD repeat and FYVE domain containing 2

            WDFY2

            1/12

            1

            match

             

            v2HS_122548

            similar to ribosomal protein S3A

            RPS3A

            4/12

            1

            pool 53

             

            v2HS_120982

            coiled coil domain containing 129

            CCDC129

            5/12

            2

            match

            59

            v2HS_111554

            interleukin 2

            IL2

            8/10

            2

            match

             

            v2HS_115544

            DEAD (Asp-Glu-Ala-Asp) box polypeptide 47

            DDX47

            2/10

            4

            match

             

            v2HS_116377

            transmemembrane protein 135

            TMEM135

            1/9

            2

            match

             

            v2HS_117064

            CD1E molecule

            CD1E

            3/9

            1

            match

             

            v2HS_119967

            LOC155004

            LOC155004

            4/9

            2

            pool 52

             

            v2HS_120757

            olfactory receptor, family 8, subfamily K, member 1

            OR8K1

            1/9

            2

            match

             

            v2HS_108647

            LOC349868

            LOC349868

            1/8

            1

            pool 79

             

            v2HS_115544

            DEAD (Asp-Glu-Ala-Asp) box polypeptide 47

            DDX47

            3/8

            4

            match

             

            v2HS_116833

            intermediate filament protein syncoilin

            SYNC

            1/8

            2

            match

            60

            v2HS_121585

            dual specificity phosphatase 3

            DUSP3

            10/11

            1

            match

             

            v2HS_117903

            glutamine rich 2

            QRICH2

            4/11

            1

            match

             

            v2HS_119967

            LOC155004

            LOC155004

            1/11

            2

            pool 52

             

            v2HS_121585

            dual specificity phosphatase 3

            DUSP3

            2/11

            1

            match

             

            v2HS_128131

            LOC351061

            LOC351061

            2/11

            1

            match

             

            v2HS_98079

            solute carrier family 22 member 3

            SLC22A3

            1/11

            2

            pool 79

             

            v2HS_119967

            LOC155004

            LOC155004

            8/10

            2

            pool 52

             

            v2HS_121585

            dual specificity phosphatase 3

            DUSP3

            2/10

            1

            match

            64

            v2HS_119967

            LOC155004

            LOC155004

            10/11

            2

            pool 52

             

            v2HS_121013

            protease serine 54

            PRSS54

            3/12

            1

            match

            66

            v2HS_117675

            latent transforming growth factor β binding protein 2/3

            LTBP2/3

            15/15

              
             

            v2HS_118530

            RNA exonuclease 1 homolog like 1

            REXO1L1

            5/13

            3

            match

             

            v2HS_117675

            latent transforming growth factor β binding protein 2/3

            LTBP2/3

            8/13

              
             

            v2HS_118530

            REX1, RNA exonuclease 1 homolog-like 2

            REXO1L1

            1/11

            3

            match

            71

            v2HS_103818

            LOC284804

            LOC284804

            2/16

            1

            pool 78

             

            v2HS_119967

            LOC155004

            LOC155004

            9/16

            2

            pool 52

             

            v2HS_97981

            LOC51152

            LOC51152

            5/16

            2

            pool 79

            72

            v2HS_119967

            LOC155004

            LOC155004

            9/10

            2

            pool 52

            74

            v2HS_119967

            LOC155004

            LOC155004

            11/12

            2

            pool 52

            77

            v2HS_119967

            LOC155004

            LOC155004

            12/12

            2

            pool 52

            78

            v2HS_94763

            paired related homeobox 1

            PRRX1

            1/8

            1

            match

             

            v2HS_99138

            solute carrier family 25

            SLC25A21

            7/8

            2

            match

             

            v2HS_102207

            transmembrane protein 63B

            TMEM63B

            1/12

            1

            match

             

            v2HS_103818

            LOC284804

            LOC284804

            4/12

            1

            match

             

            V2HS_95356

            armadillo repeat containing, X-linked 2

            ARMCX2

            2/12

            2

            match

             

            v2HS_98650

            mitochondrial ribosomal protein 63

            MRP63

            1/12

            1

            match

             

            v2HS_102155

            similar to hypothetical protein KIAA0286 (HA6800)

            KIAA0286

            1/8

            1

            match

             

            v2HS_108506

            LOC349839

            LOC349839

            1/8

            2

            match

             

            v2HS_96236

            zinc finger and BTB domain containing 1

            ZBTB1

            2/8

            1

            match

             

            v2HS_184999

            eukaryotic initiation factor 4A isoform 1

            EIF4A1

            3/10

              
             

            v2HS_96236

            zinc finger and BTB domain containing 1

            ZBTB1

            1/10

            1

            match

            79

            v2HS_105974

            LOC345597

            LOC345597

            3/11

            1

            match

             

            v2HS_184999

            eukaryotic initiation factor 4A isoform 1

            EIF4A1

            3/11

              
             

            v2HS_98079

            solute carrier family 22, member 3

            SLC22A3

            4/11

            2

            match

             

            v2HS_108647

            LOC349868

            LOC349868

            2/7

            1

            match

             

            v2HS_98079

            solute carrier family 22, member 3

            SLC22A3

            5/7

            2

            match

             

            v2HS_101943

            anthrax toxin receptor-like

            ANTXRL

            1/11

            3

            match

             

            v2HS_105093

            human solute carrier family 35, member F4

            SLC35F4

            2/11

            1

            match

             

            v2HS_106472

            LOC345713

            LOC345713

            1/11

            1

            match

             

            v2HS_107395

            LOC346589

            LOC346589

            1/11

            1

            match

             

            v2HS_91777

            nuclear receptor co-repressor N-Cor 1

            NCOR1

            4/11

            2

            match

              

            Non-protein coding RNA 282

            NCRNA00282

            1/11

              

            80

            v2HS_102441

            adenylate cyclase 1

            ADCY1

            5/11

            1

            match

             

            v2HS_130882

            glutamate receptor, metabotropic 3

            GRM3

            1/11

            3

            pool 91

             

            v2HS_97368

            yippee-like 5

            YPEL5

            2/11

            1

            match

             

            v2HS_102441

            adenylate cyclase 1

            ADCY1

            5/10

            1

            match

            82

            v2HS_184999

            eukaryotic initiation factor 4A isoform 1

            EIF4A1

            1/11

              
             

            v2HS_101845

            human prickle-like 2 (Drosophila)

            PRICKLE2

            3/11

            1

            match

             

            v2HS_109096

            LOC349975

            LOC349975

            1/11

            1

            pool 84

             

            v2HS_99423

            coiled coil domain containing 70

            CCDC70

            1/11

            1

            match

             

            v2HS_109596

              

            1/14

              
             

            v2HS_108399

            LOC349811

            LOC349811

            3/14

            1

            match

             

            v2HS_184999

            eukaryotic initiation factor 4A isoform 1

            EIF4A1

            1/14

              
             

            v2HS_93536

            proteolipid protein 1

            PLP1

            5/14

            1

            match

             

            v2HS_97017

            sterile alpha motif domain containing 4A

            SAMD4A

            1/14

              
             

            v2HS_106158

            LOC345672

            LOC345672

            3/14

            2

            match

             

            v2HS_93615

            p53

            TP53

            3/14

            1

            match

             

            v2HS_95112

            RAS p21 protein activator 4

            RASA4

            1/14

            1

            match

             

            v2HS_99525

            BCL2 like 12

            BCL2L12

            1/14

            2

            match

             

            v2HS_97017

            sterile alpha motif domain containing 4A

            SAMD4A

            4/14

              
             

            v2HS_94640

            aryl hydrocarbon receptor nuclear translocator-like

            ARNTL

            3/10

            2

            match

             

            v2HS_100174

            desmoglein 4

            DSG4

            2/10

            2

            pool 145

             

            v2HS_96026

            adnp homeobox 2

            ADNP2

            3/10

            1

            match

             

            v2HS_100819

            Rho GTPase activating protein 20

            ARHGAP20

            2/10

            1

            match

            84

            v2HS_106409

            LOC345700

            LOC345700

            1/11

            1

            match

             

            v2HS_95019

            zinc finger protein 16

            ZNF16

            9/11

            1

            match

             

            v2HS_97152

            ubiquitin-conjugating enzyme E2, J1

            UBE2J1

            1/11

            1

            match

             

            v2HS_95019

            zinc finger protein 16

            ZNF16

            10/11

            1

            match

            94

            v2HS_141495

            zinc finger protein 454

            ZNF454

            7/12

            2

            pool 96

            95

            v2HS_130457

            radial spoke head 10 homolog B (Chlamydomonas)

            RSPH10B

            6/7

            3

            match

             

            v2HS_131154

            potassium inwardly-rectifying channel, subfamily J

            KCNJ2

            1/7

            3

            match

             

            v2HS_184999

            eukaryotic initiation factor 4A isoform 1

            EIF4A1

            1/3

              

            96

             

            chromosome 9 open reading frame 123

            C9orf123

            5/13

              

            98

            v2HS_141367

            cDNA FLJ37626/LOC285500

            FLJ37626

            10/10

            2

            match

             

            v2HS_119967

            LOC155004

            LOC155004

            2/11

            2

            pool 52

             

            v2HS_133299

            insulin-like growth factor binding protein 6

            IGFBP6

            3/11

            2

            match

             

            v2HS_135564

            chromosome X open reading frame 57

            CXORF57

            5/11

            1

            match

            For each candidate the DNA pool from which it was isolated, the insert reference, target gene, frequency of isolation, as well as the number of shRNAmir constructs for that gene within the SM2 library are indicated. The last column shows if the recovered insert was a match to a hair pin in that particular pool ("match") or if it was from another pool ("pool X").

            Pool 13, 78 and 82 that produced more colonies and colonies that were larger and healthier than others, identified the following genes: Pool 13: DYNLRB1, FARSLB, PPARGC1A, TAOK1, CEND1, ABL1, LOC342404, TMEM168, SIRPG, IYD and SMCR7; Pool 78: PRRX1, SLC25A21, ARMCX2, LOC284804, LOC349839, MRP63, TMEM63B, ZBTB1, KIA0286 and EIF4A1; and Pool 82: EIF4A1, CCDC70, PRICKLE2, PLP1, SAMD4A, RASA4, TP53, ARNTL, BCL2L12, ADNP2, DSG4, LOC345672, LOC349975, LOC349811 and ARHGAP20. Moreover, pool 82, one of the pools that gave the best senescence bypass, contained the only shRNAmir targetting p53, v2HS_93615 identified in the screen, thereby internally validating it.

            Overlap of the primary candidates of the shRNA screen with microarray data for genes up-regulated upon senescence in CL3EcoR cells

            To prioritise the candidates identified from the primary screen for functional validation, they were compared to genes up-regulated upon senescence and whose expression was reversed when senescence was abrogated upon inactivation of the p53 and pRB pathways [16]; microarray expression profiling data is available from Gene Expression Omnibus database accession number GSE24810]. This identified 4 common genes, ATXN10, LAYN, LTBP2/3 and TMEM9B. The microarray expression profiling data presented in Table 2 showed that they were all up-regulated upon senescence growth arrest: ATXN10 by 1.3 fold (p-value 1 × 10-3), LAYN by 2 fold (p-value 2 × 10-4), LTBP2/3 by 2.5 (p-value 9 × 10-8), 1.7 (p-value 5 × 10-9), 1.5 (p-value 8 × 10-4) and 1.4 fold (p-value 5 × 10-5) and TMEM9B by 1.4 (p-value 1 × 10-7) and 1.3 fold (p-value 2 × 10-4) respectively. The data in Table 2 further show that up-regulation of these candidates was reversed when senescence was bypassed upon inactivation of the p53-p21 and p16-pRB pathways by silencing p53 (pRS_p53) or p21CIP1 (pRS_p21) or by sequestration of the RB family of proteins by HPV16 E7 or by expression of the dominant negative E2F-DB protein.
            Table 2

            Microarray expression profiling data for common genes

            Probe

            Symbol

            GA

            p-value

            HS

            p-value

            pRS_p53

            p-value

            pRS_p21

            p-value

            E7

            p-value

            E2F_DB

            p-value

            208832_at

            ATXN10

            0.36

            1E-03

            0.25

            3E-02

            -0.18

            2E-01

            -0.35

            2E-03

            0.02

            9E-01

            -0.23

            4E-02

            228080_at

            LAYN

            1.04

            2E-04

            0.30

            3E-01

            -0.95

            1E-03

            -0.70

            2E-02

            -0.56

            6E-02

            -0.11

            8E-01

            219922_s_at

            LTBP2/3

            1.32

            9E-08

            0.66

            5E-03

            -1.04

            2E-05

            -1.32

            3E-07

            -1.19

            1E-06

            -1.41

            4E-08

            223690_at

            LTBP2/3

            0.75

            5E-09

            -0.14

            3E-01

            -0.52

            1E-05

            -0.91

            2E-10

            -1.27

            1E-14

            -1.22

            3E-14

            204682_at

            LTBP2/3

            0.57

            8E-04

            -0.48

            8E-03

            -0.39

            4E-02

            -0.94

            1E-06

            -1.62

            3E-12

            -2.02

            4E-15

            227308_x_at

            LTBP2/3

            0.46

            5E-05

            0.03

            9E-01

            -0.43

            3E-04

            -0.57

            4E-06

            -0.28

            2E-02

            -0.55

            4E-06

            218065_s_at

            TMEM9B

            0.44

            1E-07

            -0.11

            2E-01

            -0.33

            6E-05

            -0.38

            9E-06

            -0.20

            1E-02

            -0.18

            2E-02

            222507_s_at

            TMEM9B

            0.39

            2E-04

            -0.19

            1E-01

            -0.28

            1E-02

            -0.34

            2E-03

            -0.12

            3E-01

            -0.07

            6E-01

            Log2 fold changes in gene expression in CL3EcoR cells upon senescence arrest are indicated as GA and upon shift up of control temperature insensitive HMF3S cells, from 34°C to 38°C, as HS respectively. Also indicated are the data obtained for these genes from CL3EcoR cells in which senescence had been bypassed by silencing of p53 (pRS_p53) or p21CIP1 (pRS_p21) or by inactivation of pRB using HPV16 E7 (E7) or by expression of the dominant negative E2F-DB protein.

            The identification of TMEM9B was particularly remarkable because the microarray analysis has suggested that senescence growth arrest in CL3EcoR cells is associated with activation of the NF-κB signalling pathway and TMEM9B has previously been shown to be able to activate NF-κB dependent reporter constructs [18, 19]. To determine if silencing of TMEM9B would bypass senescence, 4 GIPZ lentiviral silencing constructs (v2LHS_247318, 58957, 58958 and 58959; Thermo Scientific Open Biosystems) targetting TMEM9B were obtained, pooled and introduced into CL3EcoR cells after packaging as lentiviruses. The GIPZ lentiviral library contains the same hair pins as the retroviral library but is more stable and the constructs are packaged as lentiviruses rather than retroviruses. Lentiviral human GIPZ Lamin A/C shRNAmir (v2LHS_62719) was used as a negative control. Silencing of TMEM9B was clearly able to bypass senescence (Figure 4a). Moreover each of the constructs was individually able to overcome senescence arrest with v2LHS_58957 being the most efficient [16].
            http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-355/MediaObjects/12864_2010_3526_Fig4_HTML.jpg
            Figure 4

            Silencing targets identified from the primary screen. CL3EcoR cells were infected in triplicate with lentiviruses prepared from the indicated GIPZ shRNAmir constructs targetting TMEM9B (a), LTBP2/3 (b), ATXN10 (c) and LAYN (d). Cells resistant to 6 μg/ml puromycin were isolated, reseeded and assayed for bypass of senescence by culturing at 38°C for 3 weeks. Lentiviruses prepared from the GIPZ shRNAmir Lamin A/C (v2LHS_62719) construct were used as the negative control. For TMEM9B, the mix of constructs comprised v2LHS_247318, 58957, 58958 and 58959.

            To determine if ATXN10, LAYN and LTBP2/3 were also able to directly bypass senescence, lentivirus constructs from the GIPZ lentiviral shRNAmir library were used. The complementation assay for LTBP2/3, in Figure 4b, showed that silencing LTBP2/3 with the one available silencing construct (v2LHS_34089) clearly yielded healthy growing colonies. Silencing of ATXN10 was tested using 4 different silencing constructs v2LHS_71735, 71736, 71737 and 71740. All four constructs were able to overcome senescence and yielded more growing colonies than the Lamin A/C negative control (Figure 4c). Silencing of LAYN was tested using 2 different silencing constructs v2LHS_265009 and 118722; both constructs were able to bypass growth arrest and produced growing colonies (Figure 4d).

            Taken together our results showed that silencing of TMEM9B, ATXN10, LAYN and LTBP2/3 was able to bypass senescence in the conditionally immortal human fibroblasts.

            Discussion

            To directly identify the downstream effectors of the p53-p21 and p16-pRB pathways crucial for mediating entry into senescence, we carried out a loss-of-function RNA interference screen in the conditionally immortal HMF3A human fibroblasts. This identified 112 known genes including p53 and another 29 shRNAmirs targetting unidentified loci. Comparison of these known targets with genes up-regulated upon senescence in these cells identified 4 common genes TMEM9B, ATXN10, LAYN and LTBP2/3. Direct silencing of these common genes using lentiviral shRNAmirs bypasses senescence in the HMF3A cells. Although none of these genes has previously been linked to cellular senescence, TMEM9B has been suggested to be an upstream activator of NF-κB signalling which we have found to have a causal role in promoting senescence.

            The 112 known targets identified by the shRNA screen comprise a wide variety of genes but most importantly one of them was the only p53 shRNAmir (v2HS_93615 from pool 82) present within this library, thereby internally validating the screen. Moreover all of the primary targets were identified from single shRNAmirs even though we have subsequently shown that other shRNAmirs corresponding to these targets present within the library are able to bypass senescence. It is not clear why other shRNAmirs were not isolated in the screen; however this is exactly what has been observed previously by others such as Westbrook and colleagues [20]. Nevertheless it remains to be demonstrated which of the targets identified by the primary screen are able to bypass senescence when assayed individually.

            TMEM9B was one of the 4 genes in common between the shRNA screen and genes known to be up-regulated upon senescence in HMF3A cells [16]. Moreover expression of TMEM9B was down-regulated when senescence was bypassed upon abrogation of the p53-p21 or p16-pRB pathways. TMEM9B is a glycosylated protein that localises to lysosome membranes and partially to early endosomes. It has been shown to be a component of TNF signalling and a module shared between the interleukin-1 and Toll-like receptor pathways. It is also essential for TNF activation of both NF-κB and MAPK pathways by acting downstream of RIP1 and upstream of the MAPK and IκB kinases at the level of the TAK1 complex [19]. TMEM9B was also identified in a large scale study to identify genes activating NF-κB and MAPK signalling pathways [18]. These results are all consistent with our finding that in the conditionally immortal HMF3A cells, senescence growth arrest is associated with an activation of NF-κB signalling and suppression of this pathway bypasses senescence [16].

            The latent TGFβ-binding protein 2/3 (LTBP2/3) hair pin sequence was identified from pool 66. Up-regulation of LTBP2/3 expression upon growth arrest was reversed when senescence was overcome. LTBPs are secreted proteins initially identified through their binding to TGFβ and may be involved in their assembly, secretion and targetting [21]. LTBP2/3 in particular has been found to play an essential role in the secretion and targetting of TGFβ1 [22]. Since silencing of LTBP2/3 can bypass senescence in HMF3A cells, it suggests that LTBP2/3 may be linked with the control of cell growth and be playing a role in suppressing tumour progression perhaps through regulation of TGFβ. This is in accordance with the identification of TGFβ as a senescence-inducing factor in the human lung A549 adenocarcinoma cells [23]. It is also in accordance with several other reports suggesting that TGFβ1 is capable of inducing cellular senescence. For instance, stimulation of human diploid fibroblasts with TGFβ1 triggers the appearance of senescence associated-β-galactosidase activity and increases steady state mRNA levels of senescence associated genes including APO J, fibronectin, and M22 [2426]; both APO J (clusterin) and fibronectin are up-regulated in CL3 cells upon senescence arrest and this is reversed when senescence is bypassed [16].

            Ataxin (ATXN) 10 was slightly up-regulated upon senescence arrest which was reversed upon silencing of p53 and p21CIP1 or ectopic expression of the dominant negative E2F-DB protein. Spinocerebellar ataxia type 10 (SCA10) is a dominantly inherited disorder characterized by ataxia, seizures and anticipation caused by an intronic ATTCT pentanucleotide repeat expansion. The product of SCA10 encodes the novel protein, ATXN10, previously known as E46L, which is widely expressed in the brain and belongs to the family of armadillo repeat proteins. Although clinical features of the disease are well characterized, very little is known about ATXN10. ATXN10 knock down by RNAi has recently been shown to cause increased apoptosis in primary cerebellar cultures, implicated in SCA10 pathogenesis [27, 28]. This is in contrast to our finding that silencing of ATXN10 in HMF3A cells by four different shRNAmirs did not cause apoptosis but promoted growth and permitted a bypass of senescence.

            Layilin (LAYN) identified from pool 58 was 2 fold up-regulated upon senescence arrest, which was reversed upon abrogation of the growth arrest by inactivation of either the p53-p21 or the p16-pRB pathways. Moreover two different LAYN shRNAmirs were found to directly bypass senescence in HMF3A cells. Layilin is a widely expressed integral membrane hyaluronan receptor, originally identified as a binding partner of talin located in membrane ruffles. Talin is responsible, along with its adaptor proteins, for maintaining the cytoskeleton-membrane linkage by binding to integral membrane proteins and to the cytoskeleton. Recently layilin has been suggested to play a crucial role in lymphatic metastasis of lung carcinoma A549 cells [29].

            In addition to the genes described above, a number of other interesting genes particularly TAOK1, RAS4A and ARMCX2 were identified. TAOK1 is a micro-tubule affinity-regulating kinase required for both chromosome congression and checkpoint-induced anaphase delay [30]. It is known to activate the p38MAPK pathway through the specific phosphorylation of MKK3. This is a complex pathway responsive to stress stimuli and involved in cell differentiation and apoptosis and has been shown to have an important causative role in senescence [31]. RAS4A encodes a member of the GAP1 family of GTPase-activating proteins that have been identified to suppress the Ras/mitogen-activated protein kinase pathway in response to an elevation of Ca2+ ions. Stimuli that increase intracellular Ca2+ levels result in the translocation of this protein to the plasma membrane, where it activates Ras GTPase activity resulting in Ras being converted from the active GTP-bound state to the inactive GDP-bound state and suppression of downstream signalling [32]. ARMCX2 encodes a member of the ALEX family of proteins and may play a role in tumour suppression. This protein contains a potential N-terminal transmembrane domain and a single Armadillo repeat; armadillo repeat containing proteins are involved in development, maintenance of tissue integrity and suppressing carcinomas [33].

            Conclusions

            The RNA interference screen has identified 112 known candidate proteins including p53 and another 29 shRNAmirs targetting as yet unidentified loci. Although none of them except p53 had previously been linked to senescence or known to be downstream effectors of the p53-p21 and p16-pRB tumour suppressor pathways, directly silencing four of these candidates, TMEM9B, ATXN10, LAYN and LTBP2/3 bypassed senescence in CL3EcoR cells. It remains to be determined whether direct silencing of any of the other primary candidates will also bypass senescence. Any genes that can bypass senescence upon silencing are novel starting points for identifying the signalling networks that act downstream of p53 and pRB to induce cellular senescence. The genes/proteins identified in the screen are also potential tumour suppressors, and a mechanistic dissection of their mode of action and role in cancer will undoubtedly provide new avenues for further research.

            Methods

            Cell Culture

            CL3EcoR cells were maintained at 34°C ± 0.5°C [15]. Temperature shift experiments were performed at 38°C ± 0.5°C. Phoenix ecotropic and HEK293T cells were obtained from the ATCC and maintained at 37°C. Cells were grown in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% (v/v) heat inactivated foetal bovine serum, 2 mM glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin. All media and components were obtained from Invitrogen.

            Viral packaging and infection

            Lentiviruses were prepared according to Besnier et al. [34]. Ecotropic viruses were prepared by transfecting 10 μg of retroviral plasmid DNA into phoenix ecotropic cells by FuGENE 6 Transfection reagent (Roche), according to the manufacturer's instructions. 24 hrs post-transfection, media was changed and fresh medium added. 48 hrs post-transfection, retroviral supernatant was harvested, filtered through a 0.45 μm filter and either used immediately or frozen at -80°C. A second harvest was prepared by adding fresh media to the plates and harvesting the virus supernatant the next day.

            Cells were infected with virus supernatants for 24 hrs at 34°C. Four days post-infection, antibiotic selection was added (2 μg/ml puromycin for pRS and pSM2 retroviruses or 6 μg/ml puromycin for pGIPZ shRNAmir lentiviruses; Invitrogen). Selection of cells infected with human GIPZ lentiviral shRNAmir constructs in puromycin at 6 μg/ml, enriches for cells with higher levels of shRNAmir expression. For the senescence bypass assay, the stably transduced cells were plated at 5 × 104 cells in T-75 flasks or at 1.6 × 104 cells in T-25 flasks and incubated overnight at 34°C. Next day the medium was changed and the cells shifted to 38°C for 3 weeks. Flasks which contained more densely growing or bigger colonies were trypsinised, replated and used for extracting genomic DNA when confluent.

            Declarations

            Acknowledgements

            We are indebted to Catherine King (the UCL shRNA library core facility) for providing constructs, Gary Adamson for DNA sequencing and to Ray Young for graphics. PSJ gratefully acknowledges financial support from the Wellcome Trust (078305) and an equipment grant from the Brain Research Trust.

            Authors’ Affiliations

            (1)
            Department of Neurodegenerative Disease, UCL Institute of Neurology
            (2)
            Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research

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            This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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