Patent Application: US-201113878387-A

Abstract:
the present invention discloses a series of genes and / or proteins associated with cutaneous squamous cell carcinoma and provides polynucleotides and / or polypeptides for use in the treatment and / or prevention of cscc . the invention further relates to methods of diagnosing cscc and provides oligonucleotides / polypeptide probes and primers .

Description:
the present invention will now be described in detail with reference to the following figures which show : fig1 — cscc keratinocytes readily form tumors in scid mice with identical histology to human cscc . female scid balb / c mice were subcutaneously injected in the right flank with 1 - 4 × 10 6 tumor cells mixed with high - concentration matrigel ®( becton - dickinson , oxford , uk ). tumor volumes were measured twice a week with callipers and calculated using the formula v = π4 / 3 [( l + w )/ 4 ] 3 , were l is the length and w is the width . ( a ) representative growth of 8 separate cscc keratinocyte populations . ( b ) number of days to reach a volume of 100 mm 3 , data derived from 1 - 4 separate experiments n = 2 - 6 in each case . ( c ) h & amp ; e stained sections of a representative xenograft tumor for each of the 6 cell populations that showed measurable growth in mice ( 100 × magnification ), see also fig7 . fig2 — cscc tumor keratinocytes express altered p53 , p16 , increased myc , and increased phosphorylated stat3 but do not display features of activated ras . ( a - c and e ) western blotting of total cell lysates from cscc cells and normal primary keratinocytes ( nhk , separate donors in b ) isolated from reduction surgery or rdeb skin ( ebk ). ( d ) commercially available ras elisa showing no evidence of increased activated ras in cultured cscc populations compared with nhk or ebk . ebk rasv12 are a population of rdeb skin keratinocytes transduced with a mml - v based vector expressing oncogenic ras - v12 . hela extract = positive control provided . no pattern from any of the antibody observations correlate with tumor forming ability of scc keratinocytes , see also fig8 . fig3 — gene expression profiling can separate quiescent cscc keratinocytes and normal keratinocytes in an unsupervised manner and identifies an in vitro cscc signature consistently expressed in a range of in vivo data sets . ( a ) confluent keratinocytes are quiescent after 48 hours culture . growth rates of keratinocytes used in this study as assessed by mtt assay seeded at low density ( upper panel ) and at confluence ( lower panel ). rna was isolated between 48 and 56 hours post confluence . ( b ) clustering dendrogram of in vitro gene expression data generated in brb array tools v3 . 8 . 1 . cscc keratinocyte samples ( red box ) cluster independently of non - cscc keratinocyte samples ( blue box ), see also table s1 - s2 for pairwise comparison of in vitro samples and full cscc gene signature . ( c ) average cscc vs normal fold change for all 154 in vivo cscc genes plotted against average psoriatic skin vs non - lesional skin fold change reveals a strong correlation ( r 2 = 0 . 84 ) for the majority of genes and identifies those genes specifically differentially regulated in cscc . see also tables s5 - s6 for comprehensive gene lists and fig9 for go and additional geo analysis . fig4 — c20orf20 and plk1 knockdown inhibit cscc growth with no effect on normal human primary keratinocytes . ( a ) sccic1 keratinocytes were transfected with a sirna library to the 21 cscc specific up - regulated genes ( 3 sirna duplexes individually and pooled ) and cell viability assessed 72 hours post - transfection using the mts assay . the percentage of viable cells was calculated relative to values at time zero ( t0 ) and the difference in these values between treated samples and the non - targeting control sirna is shown . ( b ) sccrdeb2 ( scck ) keratinocytes and normal human keratinocytes ( nhk ) were transfected with sirna to c20orf20 and plk1 and cell viability determined by the mts assay . the percentage of viable cells was calculated relative to values at t0 and data shown as a percentage of the non - targeting control sirna value . ( c ) total rna ( c20orf20 ) or whole cell lysate ( plk1 ) was extracted from sccic1 keratinocytes and mrna or protein levels determined by qpcr or western blot respectively . all results shown represent mean ± sd . ** p & lt ; 0 . 01 , *** p & lt ; 0 . 001 compared with control ( n = 3 ). see also fig1 for sccrdeb2 sirna library screen and related data . fig5 — plk1 inhibition decreases cscc growth , induces g2m arrest and apoptosis with no effect on normal primary human keratinocytes . ( a ) cscc keratinocytes and nhk were treated for 72 hours with the plk1 inhibitors gw843682x ( 10 μm ) and bi2536 ( 5 μm ) and cell viability assessed by the mts assay . percentage viable cell number was calculated relative to values at t0 , where & lt ; 100 % represents a net decline in cell number and & gt ; 100 % represents a net increase in cell number . representatives of a minimum of three experiments are shown . results shown represent the mean ± sd , n = 3 . ( b ) cell cycle analysis was performed on cscc keratinocytes treated with the plk1 inhibitors gw843682x ( 10 μm ) and bi2536 ( 5 μm ) and stained for brdu and propidium iodide . results are expressed as the percentage of cells found at the g0 / g1 , s and g2 / m phases of the cell cycle 20 hours following drug treatment . results shown are the mean ± sd of 3 independent experiments . see also fig1 for cell cycle analysis after plk1 and c20orf20 sirna transfection ( c ) cscc keratinocytes were treated with either the plk1 inhibitors gw843682x ( 10 μm ) and bi2536 ( 5 μm ), or transfected with sirna to c20orf20 , plk1 and a cell death positive control sirna . cells were lysed to release their cytoplasmic contents 24 or 28 hours post - treatment respectively and lysates run in an apoptosis detection elisa . the number of cleaved nucleosomes released into the cytoplasm , indicative of apoptosis , was then quantitated . results show the fold increase in absorbance associated with increased cytoplasmic nucleosomes relative to non - targeting sirna or drug vehicle control respectively . shown is a representative experiment , with each experiment performed a minimum of three times . results are the mean ± sd , n = 3 . * p & lt ; 0 . 05 , ** p & lt ; 0 . 01 , *** p & lt ; 0 . 001 compared with control . see also fig1 for a comparison of c20orf20 sirna induced apoptosis in cscc cells and the colon carcinoma line hct116 . fig6 — plk1 inhibition and c20orf20 knockdown inhibits tumor growth in vivo . female scid balb / c mice were subcutaneously injected in the right flank with 4 × 10 6 sccic1 cells mixed with high - concentration matrigel ®. when tumors reached a volume of 100 mm 3 , animals were organised in treated and control groups . ( a ) top panel : sccic1 tumors were treated with the plk1 inhibitor , bi 2536 . the treated group ( n = 3 ) was injected into the tumor with 100 μl of bi 2536 formulated in hydrochloric acid ( 0 . 1 n ), diluted with 0 . 9 % nacl at a dose of 25 mg / kg , 3 times a week during 2 weeks . the control group ( n = 2 ) was injected with the vehicle at the same schedule . tumor volumes were measured 3 times a week with a calliper for a further 2 weeks after treatment . bottom panel : sccic1 tumors were treated with c20orf20 sirna . the treated group ( n = 3 ) was injected into the tumor with 580 pmol c20orf20 targeting sirna duplex in 100 μl pbs . the control group was injected with 580 pmol non - targeting sirna duplex in approximately 100 μl pbs . each group was treated every other day for 16 cycles . the animals were sacrificed 2 days after the last treatment . ( b ) representative h & amp ; e stained section ( top ) and keratin immunostained section ( bottom ) of control ( left ) and bi2536 - treated ( right ) sccic1 tumors after 2 weeks of treatment . a subset of animals was sacrificed at the end of the 6 cycles of treatment without waiting 2 more weeks . although no difference in tumor volume was seen at this time point between control and treated group , a histological study showed that in the bi2536 treated group , the lump consisted of mainly inert material ( keratin ) and mesenchymal infiltrating cells without any discernable sccic1 tumor cells . the control tumors contained numerous sccic1 cells ( 100 × magnification ). ( c ) representative pictures from the experiment shown in ( a ) of vehicle ( top left , tumor bisected showing both halves delineated by the dashed line ) and bi2536 - treated ( top right ) sccic1 tumors . the largest sccic1 tumor treated with c20orf20 sirna ( bottom right , tumor bisected showing both halves delineated by the dashed line ) display smaller size and are hollow in appearance compared with non - targeting sirna treated tumor ( bottom left ). both images are from the experiment shown in ( a ). fig7 — keratin staining of xenograft tumours identifies keratinocyte origin . left panel : h & amp ; e stained sections of xenograft tumors right panel : immunostaining using the anti - keratin antibody mnf 116 . 4 μm sections were immunostained using vectastain elite kit and counterstained with hematoxylin . fig8 — in vitro assays of migration vary greatly in isolated cscc keratinocyte populations and do not predict xenograft growth ( a ) migration of the cscc cell populations was assessed using the transwell assay . after 18 hrs incubation , the non - migrating cells were removed and the cells in the lower portion of the filter were stained with methylene blue and lysed with 1 % sds solution . absorbance was measured using a multiplate reader at 630 nm . the absorbance for the total number of cells seeded initially was also measured the same way but without removing the non - migrating cells . the results are expressed as a percentage of the total number of cells seeded : ( absorbance of migrating cells / absorbance of total number of cells )× 100 . results show the mean ± sd of 3 independent experiments performed in triplicate for each population . ( b ) h & amp ; e stained 4μm sections of organotypic culture . 5 × 10 5 scc cells were seeded onto a collagen / matrigel gel containing 2 × 10 5 human normal dermal fibroblasts . the organotypic cultures were maintained in keratinocytes medium at an air - liquid interface conditions . after 8 days , the gels were harvested and embedded in paraffin . ( c ) organotypic invasion was calculated for the 7 populations of cscc cells . pictures from sections immunostained with a keratin antibody ( mnf116 ) were processed with image - pro ® plus 5 . 1 software as described in materials and methods . results show the mean ± sd of 3 independent experiments performed in triplicate . ( d ) in vitro scratch wound assays were performed to assess the motility of the scc cells . confluent monolayers of mitomycin c - treated scc cells were scratched with a plastic pipette tip and cultured in keratinocyte medium . pictures were taken at oh ( left panel ) and 24 h ( right panel ). fig9 — gene ontology and metastasis expression of an incongruent gene set . ( a ) gene ontology analysis of all 154 in vivo genes and a subset of 34 of these genes whose expression is oppositely regulated in relation to control , in vitro compared with in vivo ( incongruent gene set ). ( 13 ) geo data shown for flrt3 and sox4 genes from a large dataset of normal , primary tumour , peri - lesional and metastatic , prostate cancer ( gds2546 , http :// www . ncbi . nlm . nih . gov / geo / ( barrett et al ., 2009 ). fig1 — sirna knockdown of c20orf20 , plk1 , bdkrb1 , gsg2 and prss21 inhibit growth of cscc keratinocytes . ( a ) sccrdeb2 keratinocytes were transfected with a sirna library to 21 cscc specific up - regulated genes ( 3 sirna duplexes individually and pooled ) and cell viability assessed 48 hours post - transfection using the mts assay . the percentage of viable cells was calculated relative to values at time zero ( t0 ) and the difference in these values between treated samples and the non - targeting control sirna are shown . ( b ) sccic1 keratinocytes were transfected with sirna ( 3 duplexes each gene ) to a set of genes , bdkrb1 , gsg2 and prss21 , identified in the screens in addition to c20orf20 and plk1 . the percentage of viable cells relative to values at t0 was calculated and these expressed in the graph as the percentage of control value . results show the mean ± sd , n = 3 . * p & lt ; 0 . 05 , ** p & lt ; 0 . 01 , *** p & lt ; 0 . 001 compared with control . fig1 — c20orf20 sirna induces apoptosis without cell cycle change in cscc cells . ( a ) cell cycle analysis was performed on cscc keratinocytes transfected with either c20orf20 or plk1 sirna ( pool of 3 duplexes ) and stained for brdu and propidium iodide . results are expressed as the percentage of cells found at the g0 / g1 , s and g2 / m phases of the cell cycle 24 hours following transfection . results shown are the mean ± sd of 3 independent experiments . ( b ) sccic1 keratinocytes and hct116 colorectal carcinoma cells were transfected with c20orf20 sirna and a cell death positive control sirna and apoptosis induction assessed 24 hours following transfection using a cell death detection elisa . results show the fold increase in absorbance associated with increased cytoplasmic nucleosomes relative to non - targeting sirna . shown is a representative experiment with the experiment performed twice . results are the mean ± sd , n = 3 . ( c ) sccic1 keratinocytes were transfected with sirna to either c20orf20 , tip60 or a double knockdown of c20orf20 and tip60 . cell viability was assessed 48 hours post - transfection using the mts assay . the percentage of viable cells relative to values at to was calculated . results shown are the mean ± sd n = 3 . fig1 : strategy to identify 37 cscc driver genes and subsequent sirna screen . gene signatures / groups indicated in blue are derived from the initial 435 in vitro signature ( top ) in a stepwise manner as shown . subtraction criteria to derive each subsequent signature or grouping are summarized in red . fig1 : cscc tumor and normal epidermis display contrasting c20orf20 localisation . immunofluorescence staining of paraffin embedded sections of cscc tumor xenografts and normal human skin , using a novel n - terminal peptide polyclonal antibody , reveals abundant nuclear c20orf20 localisation in tumor cells , but a striking cytoplasmic distribution in suprabasal cells of the normal epidermis . c20orf20 levels in the basal compartment appear low with sporadic nuclear expression ( indicated by arrows ). left panels sho c20orf20 staining , right panels show merged c20orf20 and dapi counterstain . magnification 600 × fig1 : c20orf20 and four additional components of the tip60 hat complex , not including the catalytic subunit , are required for cscc cell survival . ( a ) to identify potential pro - tumorigenic c20orf20 functional interactions putative binding partners were determined using intact ( embl - hdi ) and a rnai - cytotoxicity screen . depletion of seven proteins , including c20orf20 , induced significant increases in cytotoxicity compared to a non - targeting control sirna ( nt ) in all experiments ( n = 3 ) across two cscc cell populations . a cell death sirna was used as a positive control . five proteins are known components of the mammalian tip60 hat complex ( highlighted red ), while two are not associated with the tip60 complex ( blue ). interestingly , depletion of the catalytic subunit of tip60 ( green ) had little effect . data shown is 48 hours post - transfection and the mean ± sd n = 3 . fig1 : western blot showing reduction in tip60 protein levels following c20orf20 sirna treatment , indicating c20orf20 may regulate tip60 expression . all human samples were collected after informed consent and in accordance with helsinki guidelines . all animals were used in accordance with uk home office regulations after approval from the university of dundee ethics committee . primary keratinocytes were isolated and grown in the presence of a mitotically inactivated 3t3 feeder layer as described ( rheinwald and beckett , 1981 ). tumor populations were verified by snp mapping ( purdie et al ., 2007 ) or cytogenetic analysis ( cunningham et al ., 2002 ) as described . for tumorigenicity assays , 1 - 4 × 10 6 tumor cells were mixed with high concentration matrigel ® ( becton dickinson , oxford , uk ) and injected sub - cutaneously into the flanks of scid balb / c mice . for tumor treatment 4 × 10 6 sccic1 cells were used . tumors were measured by caliper and treatment began when volume reached 100 mm 3 . beta actin — mabcam 8226 ( abeam , cambridge , uk ); akt —# 9272 , phospho - akt ( ser473 )—# 9271 , erk —# 9102 , phospho erk —# 9101 , stat3 —# 9139 , phospho - stat3 ( tyr705 )—# 9131 , plk1 —# 208g4 ( cell signaling technology , inc , ca ); keratin 6 — ks6 . ka12 , desmocollin 2 —# 610120 ( progen , heidelberg , germany ); c - myc — 9e10 sc - 40 , p16 — c20 sc468 , p53 — do - 1 sc126 ( santa cruz biotechnology , inc , ca ); anti - human cytokeratin antibody mnf116 ( dakocytomation , glostrup , denmark ); ras gtpase cemi elisa kit — 52097 ( active motif , carlsbad , calif . ); ras —# 61001 18 / ras , brdu —# 347580 ( becton dickinson , franklin lakes , n . j . ); bi2536 ( selleck chemicals llc , houston , tx ), gw843682x ( sigma - aldrich , dorset , united kingdom ). all sirna were purchased from sigma except allstars hs cell death control sirna ( qiagen , crawley , uk ). the entire coding region of the p53 gene was rt - pcr amplified and sequenced as described ( bourdon et al , 2005 ). proliferation was initially calculated using the cell proliferation kit i ( mtt ) ( roche diagnostics , west sussex , uk ). subsequent viability was calculated using an mts assay ( described below ). total rna was extracted from the cells ( passage & lt ; 7 ) or frozen tissue sections and purified using the rneasy kit ( qiagen , uk ) according to the manufacturer &# 39 ; s instructions , and hybridized to a hybridize 6 — sample beadchip ( whole - genome gene expression for beadstation ), v1 arrays were used for the cell culture analysis , v2 arrays were used for the tissue analysis . cubic - spline normalized signal intensities were generated for each probe using illumina &# 39 ; s beadstudio data analysis software . data were analyzed using students t test in excel ( microsoft ). fold change scc versus normal for all public data sets analyzed were generated from normalized signal intensities available at the ncbi geo database ( http :// www . ncbi . nlm . nih . gov / geo /; edgar et al ., 2002 ) using excel . for the initial high - throughput rnai screen we used a custom library to our 21 up regulated genes containing the top 3 sirna oligonucleotides per gene as ranked by sigma . in each experiment a negative control ( mission ® sirna universal negative control # 1 , sigma ) and positive control ( allstars hs cell death control sirna , qiagen ) were used . cells were seeded in 96 - well plates at 5000 cells / well in 100 μl keratinocyte media and transfected 24 hours later with sirna ( 40 nm final concentration ) using lipofectamine ™ 2000 , invitrogen , carlsbad , calif .) diluted in opti - mem ® ( invitrogen ) according to manufacturers instructions . cell viability was assessed at 48 , 72 and 96 hours post - transfection , and a reading taken at time zero ( pre - transfection ), using the mts celltitre 96 aqueous one solution cell proliferation assay ( promega , madison , wis .) according to manufacturers instructions . absorbance readings were taken at 490 nm using a versamax microplate reader ( molecular devices , sunnyvale , calif .). for rnai transfection , cells were seeded in 6 - well plates at 2 . 5 × 10 5 cells / well and 24 hours later transfected as described above . cells were left for 16 hours then trypsinized , counted on a casy counter ( roche diagnostics ltd , west sussex , uk ) and seeded in 96 well plates at 3000 cells / well in 100 μl media . cell viability was determined using the mts assay as described above , with an absorbance reading at time of seeding ( t0 ) and readings 48 and 72 hour post transfection . for small molecule inhibitor treatment cells were seeded in 96 - well plates at 3000 cells / well and 24 hours later 100 μl fresh media containing drug at the designated final concentrations added . viability was assessed as before . apoptosis was detected using the cell death detection elisa plus ( roche diagnostics ltd , west sussex , uk ). cells were seeded in 24 - well plates at 0 . 5 × 10 5 cells / well for 24 hours and either transfected with sirna or treated with small molecule inhibitors for 24 or 16 hours respectively before collecting lysates and performing elisa as described by manufacturer . cells were rnai transfected or drug treated for designated times before brdu ( sigma ) was added at 30 μm final volume for 20 min . cells were collected and fixed by dropping 1 ml cell suspension in pbs into 3 ml ice cold ethanol while vortexing . pepsin ( sigma ) was added at 1 mg / ml in 30 mm hcl for 30 min and dna denatured with 2n hcl for 20 min . anti - brdu antibody ( becton dickinson ) diluted in pbs / 0 . 5 % tween / 0 . 5 % bsa was added for one hour followed by 30 min incubation with a fitc - sheep anti - mouse igg ( sigma ). propidium iodide ( sigma ) was added in the final wash step at a concentration of 25 μg / ml and samples analyzed using a facscan flow cytometer and cellquest software ( becton dickinson ). total rna was extracted using rna bee ( amsbio , abingdon , uk ) and rneasy columns ( qiagen ) according to manufacturers &# 39 ; instructions . 5 μg rna was incubated with random primers and m - mlv reverse transcriptase ( promega ) to generate cdna . for quantitative measurement of c20 or 120 mrna , sybr green master mix ( applied biosystems , warrington , uk ) was used with the following primers : 5 ′- attcttccattcccgaatcc - 3 ′ and 5 ′- cccaaactccctgaagatga - 3 ′ ( eurofins mwg operon , germany ). primers 5 ′- gagagcttctcagacttatcc - 3 ′ and 5 ′- gtccactgctttgatgacac - 3 ′ to ef1α were used as an internal control . pcr reactions were carried out on a miniopticon real - time pcr detection system ( bio - rad , hertfordshire , uk ) and expression calculated by the δδct method ( livak and schmittgen , 2001 ). cells were grown to confluency in a 6 - well plate in keratinocyte medium . 2 h before wounding cells were treated with mitomycin c ( 10 μg / ml ) to prevent proliferation . cells were washed with pbs and a wound was made by applying a 1000 μl plastic pipette tip across the centre of the cell sheet . cells were washed twice with pbs and incubated in keratinocyte medium . a transwell system that incorporated a polycarbonate filter membrane with a diameter of 6 . 5 mm and pore size of 8 μm ( corning , sigma - aldrich , poole , uk ) was used to assess the rate of cell migration . mitomycin c - treated cells ( 1 × 10 5 ) were suspended in 100 μl of 0 . 1 % bsa dmem / hamf12 ( 3 : 1 v / v ) and seeded in the upper chamber of the transwell insert . the lower chamber was filled with 600 μl of dmem / hamf12 ( 3 : 1 v / v ) supplemented with 5 % fbs , 0 . 4 μg / ml hydrocortisone , 5 μg / ml insulin , 10 ng / ml egf , 5 μg / ml transferrin , 8 . 4 ng / ml cholera toxin and 13 ng / ml liothyronine . following 18 h of incubation at 37 ° c ., nonmigrating cells on the upper surface of the filter were removed with a cotton swab . cells that migrated to the lower surface of the filter were stained with 1 % borax and 1 % methylene blue before being lysed with a solution of 1 % sds . absorbance was measured with a microplate spectrophotometer at 630 nm . migration rate was calculated with the following equation ( od of the migrating cells / od of total number of cells seeded )× 100 %. to prepare collagen / matrigel ® gels , 3 . 5 volumes of collagen type i ( marathon laboratory supplies , london , uk ) were mixed on ice with 3 . 5 volumes of matrigel ® ( becton - dickinson , oxford , uk ), 1 volume 10 × dmem , 1 volume fbs and 1 volume 10 % fbs dmem in which normal human fibroblasts ( nhf ) had been suspended at a concentration of 2 × 10 6 / ml . the solution was equilibrated with 1m naoh and 1 ml of this solution ( 2 × 10 ′ nhf ) was cast into wells of a 24 - well plate and allowed to polymerise for 30 min at 37 ° c . after polymerization , the gels were detached from the well with a plastic pipette tip , 1 ml of 10 % fbs dmem was added per well and gels were left overnight at 37 ° c . next day , medium was aspirated and 5 × 10 5 keratinocytes ( suspended in keratinocyte medium ) were added in a clonal cylinder ( 9 . 5 mm × 11 mm , sigma - aldrich , poole , uk ) placed on the top of each gel . the following day , gels were lifted on steel grids . sufficient keratinocyte medium was added to reach the undersurface of the gel allowing the epithelial layer to grow at an air - liquid interface . medium was changed twice a week . after 8 days , the gels were harvested , fixed in paraformaldehyde and embedded in paraffin . sections of 4 gm were immunostained with the anti - human cytokeratin antibody mnf116 ( dakocytomation , glostrup , denmark ) using vectastain elite kit ( vector laboratories , peterborough , uk ). an invasion index was calculated as previously reported ( nystrom et al ., 2005 ). briefly , digital images of keratin immunostained sections were analysed using image - pro ® plus 5 . 1 software ( media cybernetics , bethesda , md ., usa ). the digital images were converted to greyscale and immunostained areas were converted to saturated red particles using the threshold function . this was subjected to two ‘ clean - up ’ procedures : the first to remove all particles less than ten pixels in size using the “ select measurement ” function . next , any artifact was manually removed by comparing the processed image to the immunostained image . the main event representing the epidermis was removed . the saturated image was then virtually split in 4 zones of 500 pixels in its width and the lengths of invasion of the deepest event were measured in each of the zones and were used to determine the average length of invasion ( a ). the number of events ( b ) as well as the sum of the area of these events ( c ) were also calculated . the invasion index was determined by a × b × c . gene ontology was curated manually based on literature searches using pubmed at the ncbi website . primary keratinocytes derived from cutaneous squamous cell carcinoma readily form in vivo tumors with histological features of cscc in order to model human cscc without the need for genetic manipulation we isolated keratinocytes directly from fresh human tumor material as described ( rheinwald and beckett , 1981 ). because we wanted to study life threatening cscc we isolated keratinocytes from tumors which presented with metastasis derived from immuno - competent and immuno - suppressed patients ( uv induced scc ) and also tumors derived from patients with recessive dystrophic epidermolysis bullosa ( rdeb ), an inherited skin blistering disease where cscc are aggressive and frequently lead to mortality ( fine et al ., 2009 ). for comparison we used cscc keratinocytes isolated from well differentiated tumors which did not present with metastasis and from non - scc primary epidermal keratinocytes . previous studies demonstrate that tumor keratinocytes can be identified through long term proliferative capacity in vitro with retention of primary tumor genetic alterations determined through snp mapping ( purdie et al ., 2007 ). all tumor keratinocytes used in this study showed clear genetic alterations as determined by 10k or 250k snp mapping array hybridization and cytogenetic analysis ( data not shown ). 5 / 8 tumor keratinocyte populations readily formed tumors in scid mice , 1 / 8 of the populations consistently formed squamous cysts which failed to reach 100 mm 3 volume during the experiment and 2 / 8 tumor populations tested did not grow ( fig1 a ). xenograft tumors were readily recognized as human cscc except in the case of scct8 where the tumor population had pronounced spindle cell morphology ( fig1 b ); carcinosarcoma was considered although the cells displayed immunolabelling with a keratin antibody thereby indicating a diagnosis of poorly differentiated spindle cell cscc ( fig7 ). table 1 details the patient donors used for our in vitro studies . in vivo growth was not restricted to moderately or poorly differentiated tumors or those derived from patients with rdeb , as demonstrated by cscc keratinocytes derived from a well differentiated tumor ( scct2 ) and by an rdeb cscc xenograft tumor displaying features of keratoancathoma ( a variant of well differentiated cscc , sccrdeb3 , fig1 b and table 1 ). we investigated genes , proteins and pathways reported to be important in both human and mouse for the development of scc and looked for patterns which might separate xenograft tumor forming capability or patient group . p53 and p16 expression varied amongst cscc keratinocytes ( fig2 a ) and we detected p53 mutations in all 8 populations examined ( table 1 ). c - myc expression was consistently increased across all cscc compared with primary human keratinocytes as was phospho - stat3 expression ( fig2 b and c ). as we and others have previously reported lack of activating ras mutations in scc ( clark et al ., 1993 ; pourreyron et al ., 2007 ), we examined evidence for ras activation using elisa , phospho - erk and phospho - akt antibody staining . no consistent evidence of activated ras was observed in tumorigenic cultured cscc keratinocytes compared with normal primary or non - tumorigenic cscc human keratinocytes ( fig2 d and e ). in vitro assays of cscc keratinocyte migration and invasion are unable to predict tumor forming ability or patient donor in order to assess whether an in vitro assay could be a surrogate for tumor forming capacity or identify clinically aggressive cscc we compared migration and invasion using transwell migration , scratch wound migration and 3 - dimensional organotypic cultures . transwell migration varied greatly among cscc keratinocyte populations as did invasion into organotypic cultures ( fig8 ). ability to invade in organotypic assays or to migrate in transwell and scratch wound assays did not correlate with tumor forming capacity as evident comparing sccic1 , sccrdeb3 and scct8 ( fig1 a and fig8 ). comparison of gene expression with normal primary keratinocytes identifies a 435 cscc keratinocyte gene signature in culture to identify differences at the mrna level between cscc and non - scc keratinocytes we performed gene expression analysis using quiescent cultures of early passage primary cells . we chose to use confluent cultures to best mimic close cell - cell proximity of keratinocytes in vivo ( both cscc and non - scc ) and to eliminate changes in gene expression caused by divergent proliferation rates . mtt assay confirmed that at the point of rna isolation cultures were quiescent ( fig3 a ). un - supervised clustering of normalized signal intensities clearly segregated normal skin from cscc ( fig3 b ). pairwise comparison of disease state , histology of primary tumor or xenograft , or tumor forming ability , revealed the highest significant number of differentially expressed genes in this assay were identified comparing cscc with non - cscc cultures ( table s1 ). this analysis defined a 435 gene in vitro cscc signature ( table s2 ). 35 % of in vitro cscc genes are expressed concordantly across 3 independent in vivo mrna expression data sets to identify clinically relevant genes from our 435 in vitro cscc signature we analyzed the expression of probes representing each of the 435 genes in 3 separate tissue expression data sets containing primary cscc and normal skin samples . we performed our own experiment comparing rna isolated from fresh frozen cscc ( n = 9 ) and non - scc ( n = 5 ) skin samples and interrogated data from two publicly available experiments containing cscc and normal skin samples using separate array platforms ( gds2200 ( nindl et al ., 2006 ) and gse7553 ( riker et al ., 2008 ) respectively ). in agreement with recent observations that little overlap exists between gene expression profiling of cutaneous or hnscc when stringent filtering criteria are applied ( braakhuis et al ., 2010 ; van haren et al ., 2009 ), probes representing only 6 of our 435 gene signature were returned as differentially expressed genes across all three in vivo data sets based on fold change & gt ; 2 and p & lt ; 0 . 005 . however , when using a fold change of 20 % increase or decrease in expression , probes representing 154 of the 435 genes were concordantly expressed across all datasets in the three separate array platforms ( table s3 ( data not shown )) suggesting that a large proportion of the genes identified in culture are relevant to cscc pathology . we designated this 154 gene set as an “ in vivo cscc signature ” and noted that 37 of these were recognized as differentially expressed genes based on fold change & gt ; 2 and p & lt ; 0 . 005 in at least one of the three data sets ( table s4 ( data not shown )). subtraction comparison with the benign hyper - proliferation disorder psoriasis identifies 37 / 154 in vivo genes as potential drivers of cscc those genes which are differentially expressed comparing cscc and normal skin can be either a driver or a consequence of disease state . one approach to identifying tumor specific changes is to compare expression profiles with comparable benign conditions . psoriasis offers such a point of comparison as this disease , though completely benign , harbors massive hyper - proliferation , along with concomitant cell cycle activation , up - regulation of signaling pathways driving keratinocyte migration , as well as a reactive inflammatory response ( haider et al ., 2006 ). indeed , comparison of fold change psoriasis versus normal ( average of data sets gse13355 and gse14905 , ( romanowska et al ., 2010 ) with fold change cscc versus normal ( average of the three in vivo data sets in table s3 ( data not shown )) revealed a striking relationship between expression ( r 2 = 0 . 8 ) indicating that the majority of our in vivo cscc signature were dysregulated analogously in psoriasis ( fig3 c and table s5 ). of the 37 differentially expressed genes ( fold change & gt ; 2 and p & lt ; 0 . 005 ) within the in vivo cscc signature there was an even greater correlation with psoriasis ( r 2 = 0 . 94 , data not shown ) indicating that highly significant markers of cscc are shared with psoriasis . intriguingly , however , a separate 37 from the 154 in vivo cscc genes did not show similar fold change in psoriasis ( fig3 c , table s6 ) and were designated “ cscc specific ” with potential driver - like properties . 22 % of in vivo cscc genes are differentially regulated in vitro compared to normal of the 37 potential drivers of cscc only 29 were similarly differentially regulated in vitro and in vivo ; 8 genes were differentially expressed ( scc versus normal ) in an opposite manner in cultured keratinocytes compared with tissue ( table s6 ). this percentage was similar when we examined the expression of all 154 in vivo cscc genes : 34 ( 22 %) were discordantly regulated in vitro compared with in vivo ( table s5 ). gene ontology analysis demonstrated that these discordantly expressed genes were disproportionately involved in cytoskeleton or signal transduction compared with all 154 genes , suggesting that cellular context is important for their expression . we predicted that if these genes did respond to cellular context then the 8 cscc specific discordantly expressed genes may be differentially regulated in metastasis . to investigate this possibility we analyzed their expression in a dataset comparing prostate primary , peri - lesional , normal and matched metastasis ( gds 2546 , ( yu et al ., 2004 )). we noted that 4 out of 5 of the cscc specific discordantly expressed genes present in this data set demonstrated a clear difference of expression in metastatic tissue ( fig9 ). however , because of discordant expression we chose not to pursue those cscc specific genes whose in vitro expression , compared with control , did not represent the same regulation in tissue . rnai screen in cultured keratinocytes identifies plk1 and c20orf20 as genes critical for tumor cell survival as 21 of the remaining 29 cscc specific genes were up - regulated in tumor keratinocytes and cscc tissue , we screened two cscc keratinocyte populations , sccic1 and sccrdeb2 , by sirna knockdown of each gene individually with 3 separate duplex sequences and assessing cell viability by the colorometric mts assay . all 3 duplexes , individually and pooled , targeting plk1 and c20orf20 consistently reduced cell viability compared with controls in a high - throughput format ( fig4 a and fig1 ). further to this , 3 additional potential targets ( gsg2 , bdkrb1 and prss21 ) were identified for follow - on studies by demonstrating ‘ hits ’ consistently with 2 / 3 sirnas ( fig1 ). plk1 knockdown and inhibition induces g2 / m arrest and apoptosis in cscc keratinocytes with no effect on normal keratinocytes the ser / thr kinase polo - like kinase 1 ( plk1 ) is an important regulator of mitosis which is overexpressed in a number of cancers ( takai et al ., 2005 ). targeted depletion or inhibition of plk1 has been shown to cause g2 / m arrest and induction of apoptosis in tumor cells without affecting normal cells ( liu et al ., 2006 ; schmit and ahmad , 2007 ). here , both rnai - mediated depletion of plk1 and activity inhibition with the small molecule inhibitors bi2536 and gw843682x resulted in a potent reduction of cell viability in cscc cells with no effect on the growth of normal primary keratinocytes ( fig4 b and 5a ). cell cycle analysis revealed an accumulation of cells at the g2 / m phase following plk1 inhibition and depletion ( fig5 b and 11a ) and a cell death detection elisa demonstrated a substantial induction of apoptosis , through an increase in cleaved nucleosomes in the cytoplasm , following both plk1 inhibition and depletion in cscc cells ( fig5 c ). together these data correlate with results in other cancers and show that reduction or inhibition of plk1 results in the induction of apoptosis in cscc keratinocytes whilst having little effect on normal keratinocytes . c20orf20 knockdown induces apoptosis in cscc cells with no obvious cell cycle arrest and does not effect normal keratinocyte growth the chromosomal segment harboring c20orf20 has been identified as frequently amplified in both colorectal cancer ( carvalho et al ., 2009 ) and cervical cancer ( scotto et al ., 2008 ) and most recently , in parallel to our work , c20orf20 was identified as being over - expressed in colorectal cancer ( yamaguchi et al ., 2010 ). this study also showed that a reduction of c20orf20 expression through stable shrna inhibited proliferation in the colon carcinoma lines hct116 and sw480 ( demonstrated by a 10 % decrease in s phase replicating cells ). no evidence of apoptosis in response to c20drf20 depletion was observed ( yamaguchi et al ., 2010 ). we sought to investigate whether c20 orf20 knockdown yielded similar results in cscc and whether it had any effect on normal keratinocytes . c20orf20 knockdown in cscc resulted in reduced cell viability assessed by mts assay ( fig4 b ). further to this , no effects on cell proliferation were seen in normal keratinocytes ( fig4 b ), but depletion in cscc induced a significant apoptotic response in the absence of any change in cell cycle parameters ( fig5 c and 11a ), the opposite to that seen in colorectal cancer cell lines . in our hands , and using sirna , knockdown of c20orf20 in hct116 cells did not demonstrate a significant increase in apoptosis ( fig1 ), in agreement with the previous study ( yamaguchi et al ., 2010 ), indicating either a more potent effect in cscc or a different mode of action in different tumor types . as c20orf20 forms part of the tip60 complex we tested whether the effects seen with c20orf20 knockdown were mediated through the tip60 hat complex itself . to do this the tip60 catalytic subunit ( kat5 ) was depleted by sirna and cell viability assessed by mts assay . although tip60 knockdown moderately reduced the proliferation of sccic1 and sccrdeb2 it was far less potent than the depletion of c20orf20 alone . in addition , double knockdown of c20orf20 and tip60 neither positively nor negatively influenced the effect of c20orf20 ( fig1 c ). together this suggests that the increased cell death / decreased proliferation seen upon c20orf20 depletion is not mediated simply through its effect on the tip60 hat complex . in order to assess the in vivo action of plk1 inhibition and c20orf20 depletion we injected either the plk1 inhibitor bi2536 ( steegmaier et al ., 2007 ) or c20orf20 targeting sirna into established sccic1 xenograft tumors . in each case we saw direct evidence of effective tumor targeting ( fig6 ). in as little as two weeks , tumors harvested from animals treated with bi2536 showed marked reduction in the presence of tumor keratinocytes compared with vehicle controls ( fig6 b ). treatment with c20orf20 sirna reduced tumor volume over time compared with a non - targeting sirna control ( fig6 a ). the largest c20orf20 sirna treated tumors showed a marked reduction in the number of tumor keratinocytes present and were hollow in appearance ( fig6 c ). our approach to target identification has not only yielded definite therapeutic targets for cscc in the form of plk1 and c20orf20 ( fig6 ) but also suggests bdkrb1 , gsg2 , and prss21 may hold similar potential ( fig1 b ). the discovery that plk1 is overexpressed and required for survival in cscc cells is encouraging since similar observations in a number of different tumor types are linked to poor prognosis ( takai et al ., 2005 ). in agreement with our findings a recent study has shown plk1 overexpression in cscc using immunohistochemical staining of skin tissue arrays ( schmit et al ., 2009 ). here , we have demonstrated that cscc keratinocytes undergo the established hallmarks of plk1 inhibition and depletion ; mitotic arrest , inhibition of proliferation and apoptosis , and as reported previously , cell death occurs preferentially in cancer cells compared to normal cells , thus providing a potential therapeutic window ( liu et al ., 2006 ; schmit and ahmad , 2007 ). normal cells require the knockdown of p53 in addition to plk1 to invoke cell death ( liu et al ., 2006 ), and various reports suggest increased sensitivity to plk1 inhibition when p53 is defective ( degenhardt and lampkin , 2010 ; guan et al ., 2005 ). this is especially pertinent in the case of cscc as both our data ( table 1 ) and that of others show that the majority of cscc harbor p53 mutation ( giglia - mari and sarasin , 2003 ), making this disease a prime candidate for plk1 targeting . the potential of plk1 as a therapeutic target that could be fast - tracked into human trials for cscc is enhanced by the fact that a number of small molecule inhibitors are already in clinical development ( degenhardt and lampkin , 2010 ; schoffski , 2009 ). among these , the inhibitor bi2536 has progressed to phase ii trials for both hematological and solid tumor malignancies ( http :// www . clinicaltrials . gov ) and is shown here to have dramatic efficacy in treating cscc in vivo . together , these data suggest that targeting plk1 has great promise for an effective cscc therapy . our observation that knockdown of c20orf20 can induce apoptosis and reduce tumor growth in cscc highlights this gene , and the histone acetyltransferase complex ( tip60 ) which it associates with , as potential targets for therapeutic development . the evidence presented here provides a much stronger argument for their targeting in cscc than in colon cancer , as advocated by yamaguchi and colleagues ( yamaguchi et al ., 2010 ). c20orf20 depletion in cscc resulted in reduced cell viability attributable to induction of apoptosis at levels comparable with plk1 knockdown , and without any effect on the cell cycle ( fig4 b , 5 c and 11 a ). this is in contrast to the data in colorectal cancer cells where a reduction in proliferation was observed without engagement of apoptosis . in addition , normal keratinocytes , which express this gene at very low levels ( fig4 c ), remain unaffected by c20orf20 knockdown ( fig4 b ). c20orf20 was first identified as a protein capable of binding to two components of the tip60 hat complex , mrg15 and mrgx ( bertram and pereira - smith , 2001 ; cai et al ., 2003 ), and although little functional data exists on c20orf20 , potential for a role in transcriptional control and / or the dna damage response exists . mrg15 and mrgx are stable components of both hat and hdac complexes , and overexpression of c20orf20 , which specifically associates with tip60 hat , has been shown to increase their protein levels , indicating regulation of stability and / or synthesis ( hayakawa et al ., 2007 ). as a result it has been suggested that c20orf20 influences the acetylation of histones and potentially transcription factors such as p53 ( gu and roeder , 1997 ) and cmyc ( patel et al ., 2004 ), by controlling the balance of mrg proteins associated with either tip60 hat or hdac complexes ( hayakawa et al , 2007 ). in the study by yamaguchi and colleagues knockdown of another c20orf20 binding partner , brd8 , also a component of the tip60 hat complex , produced effects similar to c20orf20 depletion ( yamaguchi et al ., 2010 ). it is possible that the effects of c20orf20 are mediated through interaction with other hat complex proteins and it will be necessary to systematically knockdown these components to identify functional partners specific to cscc . because of the increased potency seen in cscc compared to colon cancer it is tempting to speculate that c20orf20 is ‘ wired ’ differently in different tumor types , leading to varied responses upon reduced expression . it should also be noted that expression at the mrna level was around 2 - fold higher in cscc cells compared to hct116 ( data not shown ), perhaps indicating a greater c20orf20 - dependent pro - survival drive in cscc than in colon carcinoma . our results also suggest that simply reducing the expression of the catalytic subunit of the tip60 complex , kat5 , does not impair the effect of c20orf20 depletion , nor does it reduce cell viability as markedly . it will be important to investigate the mode of action for c20orf20 - specific apoptotic induction to clarify the potential as a cancer target . by identifying both plk1 and c20orf20 as demonstrable cancer targets we re - enforce the notion that although cultured tumor cells may fall short of faithfully replicating the complex nature of human cancers they are nevertheless invaluable in our goal to understand and ultimately treat this disease ( masters , 2000 ; sharma et al ., 2010 ). the majority of arguments against the use of cultured cells to investigate tumor biology are based on the marked differences seen in the expression profiling of cultured cancer cells compared directly with tumor tissue ( dairkee et al ., 2004 ; perou et al ., 1999 ; ross et al ., 2000 ; welsh et al ., 2001 ). few examples exist where normal cells are included in such analysis and in these cases , expression profiles in culture , as would be expected from disparate environments , cluster separately from tissue ( perou et al ., 1999 ). analysis has been restricted to comparing all profiling , cultured and tissue samples , in the search for significant differentially expressed genes and will have also overlooked differential expression relative to controls in vitro compared with in vivo ( fig9 and table s5 ). the use of quiescent , confluent cultures represents a departure from traditional in vitro mrna expression experiments which utilize log phase growing cultures , typically considered “ healthy ” ( perou et al ., 1999 ; welsh et al ., 2001 ). this was prompted by observations that junctional complexes in keratinocytes can take 48 hours to mature in culture ( south et al ., 2003 ; wallis et al ., 2000 ) and that varying cell - cell adhesion can modulate numerous signaling cascades ( wu and bonavida , 2009 ). by using cultured material we have been able to assess gene expression in the absence of surrounding microenvironment and supported on plastic by the cells own matrix . although it is well documented that this does not reflect the situation in vivo ( creighton et al ., 2003 ; weaver et al ., 1997 ), it has enabled us to compare tumor with normal in the absence of variation resulting from tumor heterogeneity . in doing so we make the following observations : rdeb cscc keratinocytes possess similar expression profiles to other cscc in this assay , indicating common initiation and maintenance pathways and , even after using a quiescent in vitro model , many of the “ cscc specific ” genes identified are involved in cell cycle and proliferation ( bub1 , plk1 , cdc25c , wdhd1 ; table s6 ), thus in keeping with features common to all cancers ( hanahan and weinberg , 2000 ) and suggesting that dysregulation of the cell cycle is apparent even in the absence of proliferation . subtracting similar changes in psoriatic lesional skin versus normal is a method previously used ( haider et al ., 2006 ), but here we collate data from five independent experiments and define consistent changes rather than those based on stringent selection criteria . such an approach has been identified as useful for integrating data sets ( shi et al ., 2008 ). out of the 154 in vivo cscc genes , 118 were similarly regulated in psoriasis with a strong correlation ( r 2 = 0 . 8 ), suggesting that our in vivo gene signature is not derived by chance , that the 118 genes in common play important roles in both proliferative conditions , and points to the 37 “ cscc driver ” genes as being specific to tumor phenotype . of these 37 cscc specific genes , we were unable to identify commonality with pathways previously shown to be important in cscc , data which is in agreement with our biochemical analysis ( fig2 ). in summary , we have used an integrative approach including expression profiling and in vivo assays to identify novel targets in cscc . we hope this work will lead to the use of plk1 inhibitors in the treatment of cscc and to the development of targeted therapies based around the biology of c20orf20 . p & lt ; 0 . 005 indicates student t test significance between comparison groups . mod / poor vs well differentiated excludes sccrdeb3 ( no histology available ) and tumor vs non - 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