Copy Number Analysis of Tumor Samples 2 copy number ratios less than −0.1 to be affected by partial copy number loss, and markers less than −1.28 as homozygous deletions ( Beroukhim et al., 2010 Beroukhim R.

Mermel C.H.

Porter D.

Wei G.

Raychaudhuri S.

Donovan J.

Barretina J.

Boehm J.S.

Dobson J.

Urashima M.

et al. The landscape of somatic copy-number alteration across human cancers. Mermel et al., 2011 Mermel C.H.

Schumacher S.E.

Hill B.

Meyerson M.L.

Beroukhim R.

Getz G. GISTIC2.0 facilitates sensitive and confident localization of the targets of focal somatic copy-number alteration in human cancers. Using copy number data collected from 3,131 cancer samples, we considered markers with relative logcopy number ratios less than −0.1 to be affected by partial copy number loss, and markers less than −1.28 as homozygous deletions (). Copy number profiles, and the locations, lengths, and amplitudes of the amplification and deletion events underlying these profiles, were determined as previously described (). We determined whether the difference in rates of homozygous loss, hemizygous loss, or DNA methylation were significant between CYCLOPS and non-CYCLOPS genes by comparing the observed rate of the respective copy number change to the rate after permutation of gene names.

Copy Number Analysis of Cancer Cell Lines from CCLE For each sample, we created a 100 bin histogram of copy number values for all markers, and then used a 5-bin moving average to smooth this distribution. This procedure typically yielded 2-5 well-separated peaks (local maxima with height as measured from local max to surrounding local minima > 2% of genome), presumably corresponding to integer level copy loss and gains. Based on these peaks, samples were separated into one of two categories for classification. If a sample contained one peak between log 2 copy number −0.05 and 0.05, with a second peak between −0.05 and −0.4, the first peak was defined as copy neutral, and the second peak as partial copy loss. In this case, the cutoff for copy loss was set at 95% upper bound of the second peak, and the cutoff for copy neutral was set at 95% lower bound of the first peak. If there were no peak that met our height criteria within these regions in a given sample, markers < −0.4 were considered copy loss, whereas markers > −0.2 were considered copy neutral. In either case, markers that lay between our two cutoffs were left uncalled and genes with these copy numbers were excluded from further analysis. Markers with log 2 copy number ratios ≤ −1.28 were considered homozygous loss and genes with these copy numbers were also removed from further analyses. We used the Kolmogorov-Smirnov test to determine enrichment of the CYCLOPs genes identified in our original analysis among the most significant genes in our analysis of the Validation data set.

Analysis of Copy Number and Expression Correlations 50 values for 133 cancer cell lines collected from Sanger center Cancer Genome Project ( Quantized normalized expression data were obtained from the CCLE ( www.broadinstitute.org/ccle ) and TCGA ( https://tcga-data.nci.nih.gov/tcga/ ) portals. Enrichment of Pearson correlation coefficients among CYCLOPS candidates and pathways was determined by permuting gene names. A similar analysis was used to determine significance of correlation between bortezomib log ICvalues for 133 cancer cell lines collected from Sanger center Cancer Genome Project ( http://www.sanger.ac.uk/genetics/CGP/ ) portal and the expression patterns for these lines from the CCLE ( www.broadinstitute.org/ccle ).

Analysis to Determine Correlation between Gene Dependence Scores and mRNA Expression We used the ATARiS algorithm (A.T., W.C.H., and J.P.M., unpublished data) to determine gene dependencies for individual cell lines using only shRNAs that have similar effects across cell lines. ATARiS assesses gene suppression effects in each sample of multi-sample RNAi screens that include at least two RNAi reagents (siRNA or shRNA) designed to target each gene. For each gene, ATARiS generates a quantitative gene dependency score that summarizes the results of the subset of reagents targeting the gene that induce similar effects across all screened samples, under the assumption that these effects are likely due to on-target rather than off-target gene suppression. These gene dependency scores represent the determined relative level of dependency of each sample on the expression of each screened gene. In some cases, a gene may have multiple solutions based on a different subset of its shRNAs. ATARIS determined 8,280 gene dependency scores for 7,250 genes in the Achilles data set.

Competition Assays Using shRNA Constructs Neutral cells relative to cells that express shLacZ ( We found that a single lentiviral integrant expressing either shRNA-1 or shRNA-3 was sufficient to suppress PSMC2 levels in PSMC2cells relative to cells that express shLacZ ( Figure S2 B); competition assays were therefore performed at a multiplicity of infection of 1. To perform competition assays, PSMC2 shRNA-3, PSMC2 shRNA-4, and PSMC2 shLacZ in pLKO.1 were modified by inserting GFP into the puromycin cassette (BamHI/Kpn1) to yield pLKO GFP constructs that expressed these shRNAs. Cells were infected with lentivirus that contained the indicated shRNA in pLKO.1 GFP and treated for 24 hr with three fold dilutions of virus according to protocols for lentiviral infection. At 48 hr, the cells were analyzed using a BD LSR II flow cytometry system (BD Biosciences) for GFP+ cells. The well in which the viral titer resulted in approximately 50% of GFP+ cells was then cultured for 21 d. Using FACS, we analyzed these cell populations for GFP+ cells on days 7, 14, and 21 and recorded the percentage of GFP+ cells normalized to the day 0 time point.

Reagents The following antibodies were used: PSMC2 (Enzo, PW8825), PSMC1 (Enzo, PW8305), PSMC3 (Enzo, PW8770), PSMC4 (Enzo, PW8175), PSMC5 (Enzo, PW8770), PSMD5 (Thermo Fisher, PA1975), PSMD2 (Proteintech, 14748-1-AP), PSMD11 (Enzo, PW8370), β-Actin (Santa Cruz, C4), Vinculin (Sigma, V9131), Ubiquitin (Santa Cruz, P4D1), PSMB5 (Proteintech, 19178-1-AP), PSMD1 (Enzo, PW9270), PSMA1–6 (Enzo, PW8195), PSMD4 (Enzo, PW9250), GAPDH (Cell Signaling), Anti-V5/HRP (Invitrogen), p32 (Millipore - mAb 60.11). Lentiviral vectors expressing shRNAs were obtained from The RNAi Consortium (TRC) at the Broad Institute in pLKO.1 ( http://www.broadinstitute.org/rnai/public ). The shRNA, target gene, target sequence, and TRC number are as follows: PSMC2 shRNA-1, 5′ CCTGAAGGCTTTCAAGTGAAA, TRC0000007181; PSMC2 shRNA-2, 5′ GCCAGGGAGATTGGATAGAAA, TRC0000007183; PSMC2 shRNA-3, 5′ GCCTGCCTTATCTTCTTTGAT, TRC0000007184; PSMC2 shRNA-4, 5′ CCTAAGATTGACCCAACAGTT, TRC0000007185, LacZ shRNA, 5′ TGTTCGCATTATCCGAACCAT, TRCN0000231726.

Cell Culture Barretina et al., 2012 Barretina J.

Caponigro G.

Stransky N.

Venkatesan K.

Margolin A.A.

Kim S.

Wilson C.J.

Lehár J.

Kryukov G.V.

Sonkin D.

et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Liu et al., 2004 Liu J.

Yang G.

Thompson-Lanza J.A.

Glassman A.

Hayes K.

Patterson A.

Marquez R.T.

Auersperg N.

Yu Y.

Hahn W.C.

et al. A genetically defined model for human ovarian cancer. Cancer cell lines were genotyped as part of the Cancer Cell Line Encyclopedia (). The generation of immortalized ovarian surface epithelial cells was previously described (). TYKNU and HEYA8 were cultured in DMEM supplemented with 10% Fetal bovine serum and 2 mM L-Glutamine and all the remaining cell lines were cultured in RPMI 1640 supplemented with 10% Fetal bovine serum.

Lentivirus Production Moffat et al., 2006 Moffat, J., Grueneberg, D.A., Yang, X., Kim, S.Y., Kloepfer, A.M., Hinkle, G., Piqani, B., Eisenhaure, T.M., Luo, B., Grenier, J.K., et al. (2006). A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124, 1283–1298. 5 cells in a 6 well plate containing 8 μg/ml of polybrene (Sigma). Plates were centrifuged for 15 min, 1126 X g at room temperature. For selection of virally infected cells 24 hr postinfection 2 μg/ml of Puromycin (LKO.1) or 10 μg/ml of Blasticidin (LEX303) was added. Where applicable, lysate was collected and protein levels were analyzed 5 d after infection. Lentiviruses were produced for expression constructs or shRNA constructs in 293T cells cultured in DMEM with 10% FBS supplemented with 4 mM L-Glutamine using the three-vector system as described (). The virus was diluted (1:10) and added to 2 × 10cells in a 6 well plate containing 8 μg/ml of polybrene (Sigma). Plates were centrifuged for 15 min, 1126 X g at room temperature. For selection of virally infected cells 24 hr postinfection 2 μg/ml of Puromycin (LKO.1) or 10 μg/ml of Blasticidin (LEX303) was added. Where applicable, lysate was collected and protein levels were analyzed 5 d after infection.

Inducible PSMC2-Specific shRNA System Sense-antisense oligonucleotides (IDT) were annealed and then cloned into Tet-pLKO-neo (Addgene #21916) (AgeI/EcoRI) as follows: PSMC2 shRNA-2, Sense 5′CCGGGCCAGGGAGATTGGATAGAAACTCGAGTTTCTATCCAATCTCCCTGGCTTTTTG, Antisense 5′AATTCAAAAAGCCAGGGAGATTGGATAGAAACTCGAGTTTCTATCCAATCTCCCTGGC; PSMC2 shRNA-3, Sense 5′CCGGGCCTGCCTTATCTTCTTTGATCTCGAGATCAAAGAAGATAAGGCAGGCTTTTTG, Antisense 5′AATTCAAAAAGCCTGCCTTATCTTCTTTGATCTCGAGATCAAAGAAGATAAGGCAGGC; LacZ shRNA, Sense 5′CCGGTGTTCGCATTATCCGAACCATCTCGAGATGGTTCGGATAATGCGAACATTTTTG, Antisense 5′AATTCAAAAATGTTCGCATTATCCGAACCATCTCGAGATGGTTCGGATAATGCGAACA. Plasmid sequences were confirmed by sequencing. Lentiviruses generated using these constructs were used to infect either OVCAR8 or A2780 cells. Stably infected cells were selected with Geneticin (500 ug/ml) (Sigma). Cells were treated with 100 ng/ml of doxycycline unless otherwise indicated (Sigma). Beginning 4 d after the addition of doxycycline, OVCAR8 cells begin to die. Therefore, to assess the level of PSMC2 suppression in OVCAR8 cells, we collected cells 3 d after the addition of doxycycline. Because the proliferation and viability of A2780 cells, in contrast, are not affected by the addition of doxycycline, we collected cells 4 d after the addition of doxycycline at which point PSMC2 levels had achieved a new steady state. All proliferation or viability studies on both cell lines were conducted 7 d after the addition of doxycycline. Lysates from cells using this system were made in Buffer A for all immunoblots except for Ubiquitin immunoblots, which were made in Buffer B.

Ectopic V5-PSMC2 Expression PSMC2 was engineered with a V5 sequence at the N terminus and cloned into pLEX303. To minimize a second translation initiation site that used the endogenous ATG, we amplified PSMC2 using a 5′ primer that contained a sequence that would code for the V5 epitope and also mutated the endogenous methionine to a threonine. The sequence of the exogenous construct added the following amino acids to the N-terminus (MGKPIPNPLLGLDST) where the final T (Threonine) is in place of the endogenous methionine. No modifications were made to the C terminus. OVCAR8 or IOSE cells were serially infected with lentiviruses containing either pLEX303 GFP (obtained from the TRC) or pLEX303 V5-PSMC2. Repeat infections were performed with respective constructs until the expression of ectopic V5-PSMC2 (as measured by immunoblot) was comparable to the endogenous protein. OVCAR8 cells that expressed either V5-PSMC2 or GFP were infected with lentivirus that express PSMC2 shRNA-1 or shLacZ in pLKO.1. 5 d after infection, the cells were analyzed by immunoblot and 7 d after infection, in triplicate the cells were analyzed for total ATP content (Promega). Relative ATP content was normalized to the cells infected with shLacZ. Cell lines used for orthotopic xenograft tumors were engineered to express firefly luciferase. Lentiviruses made from vectors containing the luciferase gene (pLEX301) and obtained from the TRC were used to infect the indicated cell lines.

Protein Lysates All cells were harvested and pelleted in cold PBS. All subsequent procedures were performed at 4°C. For lysate generated in “Buffer A,” the cell pellet was resuspended in 10% Glycerol, 25 mM HEPES pH 7.4, 10 mM MgCl 2 , 1 mM ATP, 1 mM DTT, and phosphatase and protease inhibitors without EDTA (Roche). Sonication was performed at low intensity using a micro-tip on ice for 1 min (50% cycle). The resulting cell mixture was centrifuged at 13000 X g for 15 min at 4°C. The supernatant was collected and centrifuged at 100,000 X g for 60 min. The subsequent supernatant was collected and used in future studies as lysate. Protein amount was normalized using the Bradford reagent (Bio-Rad). For lysate generated in “Buffer B,” the cell pellet was resuspended in 20 mM Tris-HCL pH 7.5, 150 mM NaCl, 10% Glycerol, 1% Triton-X, 0.2 mM DTT, 250 μg/ml NEM supplemented with protease inhibitors (Roche) and Phosphatase Inhibitors (PhosStop -Roche). Samples were incubated for 15 min and supernatants were collected after centrifugation at 15,000 X g. Finally, for lysate generated in “RIPA,” the cell pellet was resuspended in 1X RIPA buffer (Boston Bioproducts) that was supplemented with protease inhibitors with EDTA (Roche) for 15 min. The supernatant was collected after centrifugation at 15,000 X g for 15 min. Protein lysates made in RIPA or Buffer B were normalized with THERMO BCA normalization kit (Bio-Rad), using a BSA standard curve. Lysate analysis of 26S proteasome components across ovarian cancer cell lines and PSMC2 levels following shRNA expression were generated in RIPA buffer. For all experiments that involved native analysis of the proteasome, lysate was made in Buffer A and then analyzed either in non-denaturing or denaturating (SDS loading buffer) conditions.

Purification of ComplexPSMC2 and the 26S Proteasome Continuous glycerol gradients (from 10%–40%) were made in 25 mM HEPES pH 7.4, 10 mM MgCl 2 , 1 mM ATP, 1 mM DTT. 18 mg of lysate (Buffer A) was loaded at the top of a 14 ml gradient and centrifuged for 20 hr at 195,000 x g. Fractions were removed from top of each gradient in 1 ml increments. Native PAGE described above was used to determine which fractions contained Band A or 26S proteasome and the indicated pooled fractions were then pooled and incubated with Anti-V5 agarose conjugates. Immunoprecipitates were eluted with 20 μg/ml of V5 peptide overnight at 4°C and analyzed by immunoblot.

Immunoprecipitation of ComplexPSMC2 and the 26S Proteasome Immunoprecipitation was performed by incubating fractions with 50 μl of Anti-V5 agarose conjugated beads (Sigma) and rotating for 16 hr at 4°C. Each wash step began with centrifugation at 1,000 X g for 3 min, removal of the supernatant, and resuspension of the beads in 1 ml of buffer A. Three washes were completed and the samples were then incubated in 100 μl of 20 μM V5 peptide (Sigma) in buffer A. Equal volumes were then denatured in SDS loading buffer and analyzed by immunoblot.

Determination of PSMC2 mRNA Levels Cells were harvested and pelleted in PBS. RNA was extracted from cell pellets and resuspended in Trizol Reagent (Invitrogen) according to manufacturer’s protocol. The precipitated total RNA was then resuspended in 0.1 ml of a 1x TurboDNase buffer with 2U of DNase (Ambion) and incubated at 37°C for 15 min. RNA was then purified using the QIAGEN RNEasy kit according to manufacturer’s instructions for “RNA Clean Up.” 1 μg of RNA was used to generate cDNA using the M-MLV Reverse Transcriptase kit (Invitrogen) according to the recommended protocol. PCR reactions were performed in replicates of five using SybR PCR master mix (Applied Biosystems) and Ct values were automatically determined using Applied Biosystems 7300 System software. The resulting data were normalized to housekeeping genes and analyzed using the delta-delta Ct method for fold difference between control and test samples. Unless otherwise indicated PGK1 was used as an internal control. Primers used in quantitative RT-PCR are as follows: PGK1 (5′AGAGGGAGCCAAGATTGTCA, 5′ GGTATGCCAGAAGCCACAGT), Tubulin (5′TCTGTTCGCTCAGGTCCTTT, 5′ TGTGTCCTTGCACCCAAATA), and PSMC2 (5′ TCCACCCGGTACAGGCAAGACACT, 5′ CGAGCCCCCTCACCGACGTA).

PSMC2 siRNA Experiments in A2780 Cells 5 × 105 cells were plated in 10 cm plates on Day 0. Each plate was transfected with a total of 500 pmol of siRNA and 20 μl lipofectomine RNAi Max (Invitrogen) using the manufacturer’s recommended procedure. Three pre-annealed PSMC2 siRNAs were obtained from IDT DNA and pooled in equimolar ratios: siRNA-1 (5′-GCUGUAAAUAAGGUCAUUAAGUCTT, 5′-AAGACUUAAUGACCUUAUUUACAGCUU), siRNA-2 (5′-AGAUAAUCAAUGCUGAUUCGGAGGA, 5′-UCCUCCGAAUCAGCAUUGAUUAUCUUU), siRNA-3 (5′-CCCACAUAUUUAAGAUUCACGCUCG, 5′-CGAGCGUGAAUCUUAAAUAUGUGGGUC). The concentration of the pool was varied and the difference was made up with control siRNA (Ambion). 24 hr after transfection, 2.5 × 105 viable cells (determined by trypan blue) were plated in duplicate for a six-day proliferation assay. The remaining cells were plated and harvested for lysate (RIPA) 3 d after transfection.

PSMC2 siRNA Experiments in A2780-Dox Cells 1.5 × 106 of A2780-Dox-shLacZ and A2780-Dox-shRNA-2 cells were plated in 10 cm plates on Day 0, two for each cell line. On Day 1, the media from one plate of each cell line was replaced with media containing 100 ng/ml of doxycycline. On day 2, these cells were passaged into 96 well plates at 2,500 cells/well, as well as a single 10 cm plate (to be used for RNA) for each condition and cell line. On Day 3, these cells were transfected with 0.2 mM lipofectomine RNAi Max (Invitrogen) along with siRNA at a total concentration of 20 nM, with varying levels of siRNA specific for either PSMC2 or PSMC5 using the manufacturer’s recommended procedure. PSMC2 siRNA are listed above, with additional PSMC5 siRNAs obtained from IDT DNA predesigned siRNA (Catalog # HSC.RNAI.N002803.12.1, HSC.RNAI.N002803.12.2, HSC.RNAI.N002803.12.3) and pooled in equimolar ratios. The concentration of the pool was varied and the difference was made up with control siRNA (Ambion). Proliferation was measured by cell titer glo, and qRT-PCR data were used to determine concentrations where siPSMC2 (2.5 nM) and siPSMC5 (5 nM) led to similar levels of suppression of their respective targets.

Bortezomib Sensitivity Experiments Throughout the experiment, A2780 cells engineered in the PSMC2 shRNA inducible system were either treated with vehicle or 30 ng/ml of doxycycline. Doxycycline treatment was started on Day 0. On day 4, cells were plated at 2,000 cells/ well in a 96 well plate. The following day, the cells were treated with varying concentrations of bortezomib or vehicle. Total ATP levels were measured by Cell Titer Glo (Promega) 72 hr after adding bortezomib. The data were normalized to the vehicle treated sample. Graphpad was used to determine the IC 50 by constructing a non-linear regression with a four-parameter variable slope.

Cell Cycle and Apoptosis Assays Using the PSMC2 shRNA Inducible System We cultured either A2780 or OVCAR8 cells engineered with our PSMC2 shRNA inducible system in the presence or absence of doxycycline (100 ng/ml) and collected them for analysis after 3 d. We used the BrdU Flow Kit (BD Pharmigen) according to manufacturer’s protocol to determine the percentage of cells in each phase of the cell cycle 72 hr after the addition of doxycycline. At the same time point, we independently determined the number of cells undergoing apoptosis by FACS analysis of Annexin-5 according to the manufacturers recommended procedure (Invitrogen V13241).