The online version of this article (doi:10.1186/1476-4598-11-1) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
ABH participated in the design of the experiments, carried out the RNAi screen, the confirmatory experiments, the analysis of Caspase 8 and Caspase 3/7 activation, helped generate the expression profiling data and helped draft the manuscript. JJP carried out the bioinformatic analysis, assisted with qRT-PCR analysis of the replication dependent histone genes and helped draft the manuscript. JC helped with the extraction of molecular characterization data for CASP8AP2/FLASH from genomic and transcript CRC data sets and assisted in the generation of the CASP8AP2/FLASH expression profiles. TB and FK performed the initial normalization and statistical analysis of the expression array data. KH preformed the qRT-PCR analysis of the replication dependent histone genes. SBS performed the NEFH western blot analysis. TLJ and GE assisted in the generation of the CASP8AP2/FLASH time course expression profiles. MG assisted with the RNAi screen and confirmatory experiments. MJD and TR helped define the project concept and edited the manuscript. NJC participated in the design of the experiments, helped conduct the data and bioinformatic analysis and drafted the manuscript. All authors read and approved the final manuscript
Colorectal carcinomas (CRC) carry massive genetic and transcriptional alterations that influence multiple cellular pathways. The study of proteins whose loss-of-function (LOF) alters the growth of CRC cells can be used to further understand the cellular processes cancer cells depend upon for survival.
A small-scale RNAi screen of ~400 genes conducted in SW480 CRC cells identified several candidate genes as required for the viability of CRC cells, most prominently CASP8AP2/FLASH. To understand the function of this gene in maintaining the viability of CRC cells in an unbiased manner, we generated gene specific expression profiles following RNAi. Silencing of CASP8AP2/FLASH resulted in altered expression of over 2500 genes enriched for genes associated with cellular growth and proliferation. Loss of CASP8AP2/FLASH function was significantly associated with altered transcription of the genes encoding the replication-dependent histone proteins as a result of the expression of the non-canonical polyA variants of these transcripts. Silencing of CASP8AP2/FLASH also mediated enrichment of changes in the expression of targets of the NFκB and MYC transcription factors. These findings were confirmed by whole transcriptome analysis of CASP8AP2/FLASH silenced cells at multiple time points. Finally, we identified and validated that CASP8AP2/FLASH LOF increases the expression of neurofilament heavy polypeptide (NEFH), a protein recently linked to regulation of the AKT1/ß-catenin pathway.
We have used unbiased RNAi based approaches to identify and characterize the function of CASP8AP2/FLASH, a protein not previously reported as required for cell survival. This study further defines the role CASP8AP2/FLASH plays in the regulating expression of the replication-dependent histones and shows that its LOF results in broad and reproducible effects on the transcriptome of colorectal cancer cells including the induction of expression of the recently described tumor suppressor gene NEFH.
Additional file 1: Table S1. A siRNA based RNAi screen of 418 genes representing different functional groups associated with apoptosis. Data is shown relative to siNegative transfected cells and is ranked alphabetically within the following groups; both siRNAs ≥25% decrease in cell viability, siRNA.1 ≥25% decrease in cell viability, siRNA.2 ≥25% decrease in cell viability, both siRNAs. ≤25% decease in cell viability. (PDF 137 KB)12943_2011_980_MOESM1_ESM.PDF
Additional file 2: Figure S1. Optimization, execution and follow up of a siRNA based RNAi screen of genes associated with apoptosis. (A) Confirmation of Oligofectamine (Invitrogen) transfection conditions using CTNNB1 mRNA expression as an end-point. (B) Confirmation of cell line number and transfection conditions using an siRNA corresponding to PLK1 and cell viability as an end-point. (C) Summary of siRNA screening controls. Data is shown as the viability of cells transfected by a negative control siRNA (siNeg; mean and standard deviation of 33 wells) and a positive control siRNA (siPLK1; mean and standard deviation of 11 wells). On average, silencing of PLK1 induced an over 85% decrease in cell viability. (D) Summary of screening data with gene targets ranked as shown in Additional File 1, Table S1, both siRNAs ≥25% decrease in cell viability, siRNA.1 ≥25% decrease in cell viability, siRNA.2 ≥25% decrease in cell viability, both siRNAs. ≤25% decease in cell viability. Closed circles indicate data for siRNA and open circles data for siRNA 2. (E) Two different siRNAs corresponding to 45 genes induced a 25% or greater reduction in the viability of SW480 cells compared to siNegative-transfected cells. Data is expressed relative to siNegative-transfected cells; gene targets are ranked alphabetically. Genes chosen for initial validation are marked with *. (F) Reproducibility of the effects of silencing selected genes identified as reducing the viability of SW480 cells. Data is shown as the mean ± SD of three independent transfections for each siRNA targeting the fifteen genes of interest normalized to the values from negative control siRNA transfected cells. (PDF 633 KB)
Additional file 3: Table S2. The sequences of gene specific siRNAs. (PDF 60 KB)12943_2011_980_MOESM3_ESM.PDF
Additional file 4: Table S3. The sequences of gene specific PCR primers. (PDF 52 KB)12943_2011_980_MOESM4_ESM.PDF
Additional file 5: Table S4. Transcription factor data sets used for gene set enrichment analysis. (PDF 73 KB)12943_2011_980_MOESM5_ESM.PDF
Additional file 6: Figure S2. The development of a CASP8AP2/FLASH RNAi signature in SW480 cells. (A) Correlation of the fold change in expression seen following silencing with two different CASP8AP2/FLASH siRNAs. Each black circle corresponds to a single probe. A red circle indicates the probe corresponding to CASP8AP2/FLASH. There was a high concordance between the effects mediated by both siRNAs targeting CASP8AP2/FLASH, only two probes showed discordant changes with respect to the direction of change in that one siRNA induced an increase in expression, while the second siRNA induced a decrease in expression. (B) Within the very large CASP8AP2/FLASH expression profile we identified only one transcript (ABLIM2) that showed a potential mismatch with both CASP8AP2 siRNAs. The probe corresponding to ABLIM2 was ranked as the 349th down-regulate/d probe (out of a total of 1487 downregulated probes; ranked ~Log2 -2.0 to Log2 -0.6 fold change) within the CASP8AP2/FLASH expression profile. As this change is likely to have had only a minimal affect on the CASP8AP2/FLASH expression profile as a whole the data for this gene was retained within our subsequent analysis. (PDF 674 KB)12943_2011_980_MOESM6_ESM.PDF
Additional file 7: Table S5. CASP8AP2/FLASH RNAi signature in SW480 cells. Probes that showed a significant fold change (> Log2 ± 0.6, q < 0.05) in CASP8AP2/FLASH silenced cells compared to siNeg transfected SW480 cells are listed, ranked by the fold change seen in siCASP8AP2.3-silenced cells. The probe corresponding to CASP8AP2/FLASH is highlighted in grey. The columns show: (1) The gene sequence reference, (2) the gene symbol, (3) the chromosomal position of the Agilent array probe, (4) the Agilent array probe identifier, (5) fold change siNeg vs. siCASP8AP2.3 Log2 transformed, (6) fold change siNeg vs. siCASP8AP2.6 Log2 transformed, (7) FDR (q-value), siNeg vs. siCASP8AP2.3 and (8) FDR (q-value), siNeg vs. siCASP8AP2.6. (PDF 559 KB)12943_2011_980_MOESM7_ESM.PDF
Additional file 8: Table S6. Significant gene ontology categories within CASP8AP2/FLASH RNAi signatures. (PDF 65 KB)12943_2011_980_MOESM8_ESM.PDF
Additional file 9: Figure S3. The top three knowledge-based gene networks of genes deregulated as a consequence of silencing CASP8AP2/FLASH were generated using Ingenuity pathway analysis tools. A maximum of 70 molecules was used for the generation of networks. Solid edges between gene nodes represent direct interactions and dashed lines represent indirect interactions. Red symbols indicate upregulated genes; Green symbols indicate downregulated genes respectively. The number of molecules in each network is stated, as is a score generated by IPA as an estimate of connectivity. (PDF 669 KB)12943_2011_980_MOESM9_ESM.PDF
Additional file 10: Figure S4. The development of CASP8AP2/FLASH RNAi signatures in SW480 cells over time. (A) Reduction in CAPS8AP2/FLASH mRNA measured by (i) qRT-PCR and (ii) array analysis and cell viability in SW480 cells over time. All data is normalized to the levels of CAPS8AP2/FLASH and viability observed in siNeg transfected SW480 cells. Correlation of the fold change in expression seen following silencing with three different CASP8AP2/FLASH siRNAs at (B) 48 hours and (C) 72 hours compared to siNeg transfected SW480 cells. Each black circle corresponds to a single probe. A red circle indicates the probe corresponding to CASP8AP2/FLASH. Using the data obtained at 48 and 72 hours we observed no off-target sequence alignments between transcripts showing a decrease in expression for all three siRNAs and the sequences of the three different CASP8AP2/FLASH siRNAs though nine genes showed off-target alignments with two of three siRNAs (data not shown). To assess the reproducibility of our initial findings we examined the expression of genes that previously showed differential expression following transfection of siCASP8AP2.3 and siCASP8AP2.6 siRNAs, with data obtained at the same time point (72 hours) with same siRNAs as part of this follow up study. The data for ~2000 probes were compared. (D) Correlation of the fold change in expression seen in two different experiments in which CASP8AP2/FLASH has been silenced for 72 hours. Each black circle corresponds to a single probe. (PDF 4 MB)12943_2011_980_MOESM10_ESM.PDF
Additional file 11: Table S7. Changes in gene expression over time following silencing of CASP8AP2/FLASH. Data is shown for the 2820 probes that measured a significant change in expression in SW480 cells transfected with each of the three CASP8AP2/FLASH siRNAs at 72 hours and the data for the same probes at 48, 24 and 10 hours post siRNA transfection. The median fold change for each probe is shown as well as the data for each of the three siRNAs; data is ranked on the median value at 72 hours. Fold changes ≥ Log2 -0.6 and q < 0.05 matched to any probe showing a decreased level of expression are marked in green and fold changes of ≥0.6 and q < 0.05 matched to any probe showing an increased level of expression are marked in red. The probe corresponding to CASP8AP2/FLASH is marked is highlighted in yellow. (PDF 1 MB)12943_2011_980_MOESM11_ESM.PDF
Additional file 12: Table S8. Changes in histone transcript levels at multiple time points following silencing of CASP8AP2/FLASH. (PDF 124 KB)12943_2011_980_MOESM12_ESM.PDF
Additional file 13: Figure S5. TNF focused knowledge-based gene networks of genes deregulated as following silencing of CASP8AP2/FLASH. TNF network at (A) 48-hours, and (B) 72-hours generated using Ingenuity pathway analysis tools. A maximum of 70 molecules was used for the generation of networks. Solid edges between gene nodes represent direct interactions and dashed lines represent indirect interactions. Red symbols indicate upregulated genes; Green symbols indicate downregulated genes respectively. The number of molecules in each network is stated, as is a score generated by IPA as an estimate of connectivity. (PDF 487 KB)12943_2011_980_MOESM13_ESM.PDF
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- Systems-wide RNAi analysis of CASP8AP2/FLASH shows transcriptional deregulation of the replication-dependent histone genes and extensive effects on the transcriptome of colorectal cancer cells
Amanda B Hummon
Jason J Pitt
Susan B Skube
Tamara L Jones
Michael J Difilippantonio
Natasha J Caplen
- BioMed Central
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