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
© Hummon et al; licensee BioMed Central Ltd. 2012
Received: 29 June 2011
Accepted: 4 January 2012
Published: 4 January 2012
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.
KeywordsCASP8AP2 FLASH RNAi screening RNAi analysis siRNA replication-dependent histone transcripts
Cancer cells are characterized by changes in proteins that favor cell survival and proliferation, including down-regulation or de-activation of pro-apoptotic factors and cell cycle regulators, and up-regulation or activation of anti-apoptotic factors including kinases and growth factors. Targeting of specific proteins to overcome or bypass this suppression of cell death and enhancement of proliferation is a major approach for the development of anti-cancer therapies. Like all cancers, colorectal cancer is marked by genomic aberrations and transcriptional deregulation that affect multiple cellular pathways . The characterization of proteins whose function alters the underlying molecular features of CRC has the potential to identify new therapeutic strategies for CRC. Gene-specific loss-of-function (LOF) analysis, through the application of RNA interference (RNAi) based technologies, is increasingly being used to probe the role of a particular protein in a specific cellular context .
We have recently used RNAi based LOF approaches to validate the functional dependence of colorectal cancer cells on genes identified as over-expressed in CRC . Loss-of-function analysis via RNAi can also be used to identify proteins required for the survival of CRC cells that show no significant genomic or transcriptional changes. Alterations in apoptosis and related survival mechanisms contribute to both the development of CRC, and the response to treatment . For example, colorectal tumors often show increased expression of members of the anti-apoptotic BCL family including BCL2, mutations in the tumor suppressor TP53, and defects in several pathways related to inflammation including the COX2, TGF-ß, and NFκB pathways. It is likely that many less well-characterized proteins related to cell survival alter the growth of CRC cells. Identification of such genes could though give further insight into the molecular changes underlying CRC, and thus the development of new treatment strategies. To assess the feasibility of identifying proteins whose function has not previously been identified as essential for the survival of colorectal cancer cells we conducted a small-scale RNAi screen of ~400 genes in CRC cells. The gene targets were focused on proteins associated with cell survival, with an emphasis on regulators, and effectors of apoptosis. One of the candidate genes most prominently required for the survival of CRC cells was the gene encoding Caspase-8-Associated Protein 2 or FLICE-associated Huge Protein (CASP8AP2/FLASH). Subsequent whole transcriptome profiling showed that CASP8AP2/FLASH LOF has wide-ranging, and specific, effects on gene expression including deregulated expression of the replication-dependent histone genes.
Cell Culture and siRNA transfections
SW480 (an aneuploid, mismatch-repair proficient, colon adenocarcinoma cell line), SW837 (an aneuploid, mismatch repair proficient, rectal adenocarcinoma cell line), and SW48 (a mismatch-repair deficient, diploid, colon adenocarcinoma cell line) cells were obtained from ATCC (Manassas, VA) and were maintained in RPMI (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS, Invitrogen), supplemented with L-Glutamine and penicillin/streptomycin, at 37°C in a humidified atmosphere containing 5% CO2. Cells were passaged every four to five days. The choice of SW480 as the cell line for the RNAi screen was based on characteristics of the spectral karyotype that recapitulate chromosomal aberrations commonly observed in colorectal cancer . The synthetic siRNA based RNAi screen was performed using the Human Apoptosis Set Library (Qiagen, Valencia, CA) arrayed in a total of eleven 96 well plates, one siRNA per well. Gene targets were selected based on searches of the PANTHER (Protein ANalysis THrough Evolutionary Relationships) Classification System, the Gene Ontology database, and PubMed resources (E. Lader, Qiagen, Personnel communication). See Additional file 1, Table S1 for list of genes targeted and siRNA target sequences. Genes targeted included known regulators and effectors of apoptosis, proteins with less defined functions in cell survival and apoptosis and a few proteins with no known direct function linked to apoptosis so all functional groups were represented. We have previously determined successful transfection conditions of siRNAs into SW480 cells using the Oligofectamine transfection reagent (Invitrogen) . We confirmed these conditions for this study by examining the silencing of the CTNNB1 gene at an mRNA level (Additional file 2, Figure S1A) and the viability of SW480 cells following silencing of Polo-like kinase 1 (PLK1) (Additional file 2, Figure S1B). The RNAi screen was conducted as follows; transfections were performed by pre-complexing siRNA (2 pmol) with 0.6 μl Oligofectamine lipid transfection reagent (Invitrogen) in 50 μL of serum free RPMI in individual plate wells for 30 min at ambient temperature. Next, SW480 cells (7,000) were added in 50 μL RPMI supplemented with 20% FBS to yield a final concentration of 20 nM siRNA in RPMI, 10% FBS. This final mixture was incubated at ambient temperature for 1 hour before being placed at 37°C in a humidified atmosphere containing 5% CO2. After 72 hours cell viability was assayed (Cell Titer Blue Reagent, Promega, Madison, WI). As this was a relatively small-scale siRNA screen, the viability of SW480 cells following siRNA transfection was expressed relative to the average viability for cells transfected with a negative control siRNA - AllStar Negative Control siRNA (siNeg) (Qiagen Inc.) (n = 33) (Additional file 2, Figure S1C). A siRNA corresponding to PLK1 was used as a positive control on every plate (see Additional file 3, Table S2 for sequence); on average this induced a decrease in the viability of SW480 cells of greater than 85% (n = 11) (Additional file 2, Figure S1C). For gene specific analysis SW480, SW48, and SW837 cells were transfected in 96 well plates as above using 15,000 SW48 cells, 10,000 SW837 cells, and 5,000 SW480 cells per well. See Additional file 3, Table S2 for the sequences of gene specific siRNAs. Transfections for whole transcriptome analysis were performed in triplicate in 6-well plates, using 2.1 × 105 SW480 cells and 1.8 μL Oligofectamine per well.
Quantitative reverse transcription-PCR
Total RNA was isolated from cells using the RNeasy Mini kit (Qiagen) and mRNA reverse transcribed into cDNA using random hexamer primers or oligo dT and reverse transcriptase (Invitrogen SuperScriptIII). Gene expression levels for CASP8AP2/FLASH and NUP62 were assayed by quantitative reverse transcription-PCR (qRT-PCR) using Power SYBR Green technology (Applied Biosystems, Inc., Foster City, CA) and were normalized to the expression of YWHAZ. For each RT-PCR reaction, 300 ng cDNA was used. PCR was performed using default variables of the Applied Biosystems' Prism 7000 sequence detector, except for a total reaction volume of 25 μL. Primers were obtained from Operon Technologies, Inc. (Huntsville, AL) or Invitrogen. Gene expression levels for HIST1H2BD were determined by oligo-dT or random hexamer primed synthesis of cDNA (Invitrogen), and real-time PCR amplification in i-cycler (Bio-Rad) based conditions with SYBR green. HIST1H2BD expression levels were normalized to the expression of ß-actin. See Additional file 4, Table S3 for all primer sequences.
Branched DNA assay
Quantigene Probes (Panomics, Fremont, CA) corresponding to CASP8AP2/FLASH were used to directly measure gene specific mRNA levels (normalized to human cyclophilin B (PPIB) expression) in cell lysates as previously described .
Whole transcriptome analysis
RNA samples were quantified and assessed for quality on a Bioanalyzer (Agilent, Santa Clara, CA) and only samples with an RIN value > 8 were hybridized. For microarray analysis, 700 ng RNA was converted to cRNA using an oligo dT primer, labeled with Cy3, and subjected to mono-channel hybridization onto a 4 × 44K Whole Human Genome Microarray per the manufacturers instructions (Agilent). Microarrays were washed and processed using an Agilent G2565BA scanner. Data were quality controlled and extracted using Agilent Technologies' Feature Extraction (version 9.1). Microarray expression data are available at the Gene Expression Omnibus (GEO) accession GSE29405 and are in accordance with MIAME guidelines.
Tumor data: Array CGH and mRNA
Caspase 8 and Caspase 3/7 assays
The activation of Caspase 8 and Caspase 3/7 were measured using an Ac-LETD-pNA caspase-8 substrate (Caspase-Glo 8 Assay, Promega) and a DEVD peptide substrate (Caspase-Glo 3/7 Assay, Promega) respectively, following the manufacturers instructions. Plates were measured with a Victor luminometer (Perkin Elmer) at 60 min after addition of the Caspase 8 reagents and 90 min after the addition of the Caspase 3/7 reagents.
Western blot analysis
Transfections for Western blot analyses were performed in 6-well plates, using 1.8 × 105 SW480 cells and 10.5 μL RNAiMax per well. Whole cell lysates were harvested from SW480 cells 72 hours post transfection with siCASP8AP2.3 and siCASP8AP2.6 according to the Complete Lysis-M Reagent Kit (Roche, Indianapolis, IN). Reduction of CASP8AP2/FLASH mRNA was assessed by RT-PCR from replicate samples. Soluble protein fractions were run on a 10% SDS-PAGE gel and transferred to nitrocellulose membrane for 1 hour at 12 V with an additional 30 minutes at 24 V. The membrane was incubated with α-actin clone C4 (1:1000 dilution) (Millipore, Temecula, CA) and α-200 kD Neurofilament Heavy - Neuronal Marker (1:1000 dilution) (Abcam, Cambridge, MA) in 10% milk buffer in 1× PBS on a rocking platform overnight at 4 degrees. The membrane was washed 3 times for 10 minutes in 10% milk buffer in 1× PBS and incubated with HRP α-mouse (1:10,000 dilution) (Jackson ImmunoResearch, West Grove, PA) for 1 hour on a rocking platform at room temperature. The membrane was washed as before, rinsed with DI water and incubated with SuperSignal West Pico Chemiluminescent Substrate (Pierce/Thermo Fisher Scientific Rockford, IL) according the protocol. Kodak BioMax Light Film (Carestream Health, Woodbridge, CT) was used to expose the membrane for varying amounts of time.
Bioinformatic and statistical analysis
Standard statistical analyses were conducted in Excel. Comparisons of functional effects of gene silencing were assessed using the t-test (unequal variance); a p value of ≤0.05 was considered significant. Comparison of the fold change in different gene expression data sets was assessed using a Pearson's correlation.
Gene expression microarray data
Gene expression array signal intensities were Log2 transformed and quantile normalized using the R statistical computing software and the package Limma. Differential expression between groups was assessed using an empirical Bayes method for the moderated T-statistics; all probes were corrected for multiple testing using Benjamini and Hochberg's false discovery rate (FDR) [8–11]. In order to be considered differentially expressed, probes had to have an average Log2 fold change of ± 0.6 (approximately 1.5 fold change on a linear scale) and a q-value (FDR) < 0.05. In addition, probes needed to show this significant fold change in the same direction for both siRNAs targeting CASP8AP2/FLASH in the initial analysis and for all three siRNAs in the follow up study.
The time course study also included a comparison of siNeg transfected cells and untransfected cells. Any probes showing a significant differential expression (q-value < 0.05) in the siNeg cells versus untransfected SW480 cells were filtered from data used for analysis of gene ontologies, pathway analysis and gene set enrichment analysis. Of the ~44,000 probes examined this resulted in the removal of just 124 probes from the 24-hour data set, 820 probes at 48 hours, and ~1500 probes at 72 hours.
To determine if any genes exhibiting decreased expression represented interactions between the transfected siRNA and non-targeted transcripts, we assessed the alignment of each siRNA sequence with the 3'UTR sequence of all of the downregulated genes obtained following transfection of each siRNA. The open-access miRanda application http://www.microrna.org was used to perform alignment predictions between each siRNA sequence and the 3'UTR sequence for all genes that showed decreased expression with each siRNA. We then examined in detail the non-targeted genes that were downregulated by two or more siRNAs that showed a potential miRNA-like mismatch alignment with a sequence within the 3'UTR of the non-targeted gene.
One-way hierarchical clustering (average linkage) of the gene profiles was conducted on the expression data using JMP 8.0 (SAS, Cary, NC).
Gene expression microarray data analysis: Histone genes
Histone transcripts show altered levels following silencing of CASP8AP2/FLASH
CASP8AP2/FLASH SILENCED SW480 cells
Published HeLa data
Fold change (Log 2 ) siCASP8AP2.3
Fold change (Log 2 ) siCASP8AP2.6
Narita et al
Shepard et al PAS seq #
chr1 - 148098952-1
chr1-148122634-6, chr-148124269-1, chr1-148124357-1
ch6-26279217-1, ch6-26279551-20, chr6-26266834-7
Ingenuity Pathway Analysis and Gene Set Enrichment Analysis (GSEA)
Differentially expressed genes (see criteria above) were analyzed through the use of IPA (Ingenuity® Systems, http://www.ingenuity.com). Each dataset was divided into up- and downregulated genes respectively, and then was subjected to gene function (gene ontology) analysis. Enrichment of functional groups within gene sets was calculated using a one-tailed Fisher's exact test. Functional groups with a Benjamini-Hochberg (B-H) corrected p-value of < 0.05 were considered significant. Knowledge-based gene networks were also generated using IPA tools. A maximum network size of 70 molecules was used. In order to condense datasets for Gene Set Enrichment Analysis , the median value of each probe (using all siRNA transfections) was normalized to the median value of the same probe in the mock transfections (siNeg). When two or more probes mapped to a single gene, the median normalized expression value was taken so that each gene mapped to a single expression value. GSEA was run using default parameters http://www.broadinstitute.org/gsea/index.jsp. The resulting landscape plots were analyzed for peaks in the tails of the ranked gene lists. FDR q-values < 0.05 were considered significant.
Transcription factor target gene datasets
Transcription factor target gene lists were derived from ChIP-Chip, ChiP-PET, or ChIP-seq data curated from peer-reviewed literature (Additional file 5, Table S4). In certain instances, other biological assays and techniques such as gene expression and quantitative ChIP were used in conjunction with ChIP-Chip, ChIP-PET, or ChIP-Seq in order to derive higher-confidence lists. NFκB target genes were taken from http://www.bu.edu/nf-kb/gene-resources/target-genes/ (complied December 2009), which is a compilation of downstream NFkB targets reported in peer-reviewed literature. All gene lists were cross-referenced to the Ingenuity Knowledge Base as well as NCBI Entrez Gene. Genes that could not be mapped to these databases by their published or associated identifiers were excluded.
Results and Discussion
RNAi screening in the colorectal cancer cell line SW480 identifies CASP8AP2/FLASH as required for cell viability
As the silencing of CASP8AP2/FLASH showed the most consistent effect on the survival of CRC cells and has not been studied previously in the context of colorectal cancer we chose to examine this gene in further detail. CASP8AP2/FLASH was initially discovered as involved in the binding of the FAS-associated adaptor protein (FADD), and procaspase-8 . Ligand binding of FAS to the FAS cell surface receptor relays the extrinsic apoptotic death signal through recruitment of FADD, procaspase-8 activation, and formation of the death-inducing signaling complex (DISC). A number of studies have supported the interaction of CASP8AP2/FLASH with FADD and as a regulator for activation of CASPASE 8  and thus apoptosis . However, other studies have challenged the pro-apoptotic role of CASP8AP2/FLASH  and there is increasing evidence that the large CASP8AP2/FLASH protein (222 KDa) has multiple functions. A previous RNAi screen conducted in HeLa cells identified CASP8AP2/FLASH as essential for cell division  and other studies have linked it to NFκB signaling [20, 21], activation of MYB [22, 23], S-phase progression [24–26], and an involvement in histone biology. Evidence for the involvement of CASP8AP2/FLASH in histone biology includes its cellular location in nuclear organelles found adjacent to histone genes and biochemical studies linking it to histone transcript processing [24, 27–32].
Molecular characteristics of CASP8AP2/FLASH in colorectal cancer
Silencing of CASP8AP2/FLASH activates apoptosis associated markers
The activation of markers of apoptosis can result from alterations in many different cellular processes. Further, because CASP8AP2/FLASH has been linked to multiple functions we chose to use a systems-wide unbiased approach to investigate the principal downstream effects that perturbation of CASP8AP2/FLASH function has on CRC cells.
The CASP8AP2/FLASH loss-of-function induces changes in the expression of over two thousands genes
We first examined genes related to the primary annotated function of CASP8AP2/FLASH in the regulation of the extrinsic apoptotic pathway. While we observed the functional activation of caspase proteins in SW480 cells following the silencing of CASP8AP2/FLASH, at a transcriptional level only CASP7 expression was increased (~2-fold linear increase). The extrinsic apoptotic associated proteins FAS and FASLG were both modestly upregulated at transcriptional level, but there was no other obvious link to this function of CASP8AP2/FLASH. Because of the lack of a clears association with changes in the expression of proteins associated with the extrinsic apoptotic we next looked for the enrichment of genes associated with specific functional ontologies so as to assess the broad biological processes perturbed as a result of CASP8AP2/FLASH LOF. No ontology categories where enriched within the genes that were downregulated following silencing of CASP8AP2/FLASH. Many ontologies were though associated with the genes that were upregulated following CASP8AP2/FLASH LOF, including the cancer, cellular growth and proliferation, cell death and gastrointestinal disease ontologies (Figure 4B and Additional file 8, Table S6). Genes associated with the cancer, cell growth and proliferation, and cell death ontologies, included the upregulation of several cyclin-dependent kinase inhibitors, specifically CDKN1A (p21; ~2.5-fold linear increase), CDKN1C (p57Kip2; ~5-fold linear increase), CDKN2B (p15; ~2-fold linear increase), and CDKN2D (p19; ~2-fold linear increase). These cyclin-dependent kinase inhibitors are all critical negative regulators of cell cycle. Their up-regulation following silencing of CASP8AP2/FLASH could therefore be of relevance to the reduction in cell viability observed in CASP8AP2/FLASH silenced CRC cells.
As another approach to identify critical cellular networks perturbed as a result of CASP8AP2/FLASH LOF we used Gene Set Enrichment analysis (GSEA) to identify enrichment for the targets of specific transcription factors associated with CRC within the large CASP8AP2/FLASH RNAi signature. NFκB targets were significantly enriched within our list of genes upregulated after silencing of CASP8AP2/FLASH. Activation of NFκB is most frequently associated with an inhibition of apoptosis and constitutive activation of NFκB has been linked to CRC. Previous studies of CASP8AP2/FLASH function have shown that inhibition of CASP8AP2/FLASH expression suppressed TNFα induced activation of NFκB [20, 21]. Due to correction for multiple testing no specific canonical pathways were identified as perturbed by LOF of CASP8AP2/FLASH, but dense knowledge-based networks focused on TGFß, GRB2, and TNF were generated from the CASP8AP2/FLASH RNAi signature (Additional file 9, Figure S3). This TNF-centered network within the CASP8AP2/FLASH RNAi signature (Additional file 9, Figure S3, network 3) suggests that activation of TNF related signaling following silencing of CASP8AP2/FLASH may be contributing, at least in part, to the further up-regulation of the expression of NFκB target genes in SW480 cells.
MYC, a well-known proto-oncogene, is critical to the proliferation and survival of many cancers . Genomic copy number gains of MYC is observed in some 50% of colorectal tumors and as a target of ß-catenin/TCF7L2 MYC expression is frequently deregulated in CRC . Deregulated MYC expression can lead to activation of some genes and repression of other. Interestingly, we observed that after CASP8AP2/FLASH LOF, genes that are activated by MYC were repressed, while conversely genes repressed by MYC were activated (Figure 4C). This indicates that the transcriptional activity of MYC is reduced in the absence of CASP8AP2/FLASH. CASP8AP2/FLASH LOF did not directly induce differential expression of MYC suggesting that this is a secondary effect perhaps as a result of changes in proteins responsible for the post-translational modification of MYC that also regulate MYC activity .
The clearest LOF molecular phenotype that could be related to CASP8AP2/FLASH silencing was, however, alterations in the expression of the histone genes.
The CASP8AP2/FLASH RNAi signature shows broad deregulation of histone gene transcription
It is important to note that the cRNA used in this study was generated using an oligo dT primer, selective for polyadenylated transcripts, and thus the changes in the levels of the replication dependent histone transcripts would appear to reflect significant alterations in the levels of the less well defined polyadenylated versions of these histone transcripts. The detection of polyadenylated replication-dependent histone transcripts has been noted previously following silencing in HeLa cells of two other replication dependent histone 3' end processing proteins: negative elongation factor (NELF) and the cap binding complex (CBC) . Following silencing of either NELF or CBC, Nariata and co-workers, reported increases in the levels of twelve histone genes by array analysis, also following oligo dT primer selection. Examination of the expression of two of these histone genes, HIST1HC and HIST2H2AA, in detail confirmed that this result was due to the induction of the expression of polyadenylated transcript variants corresponding to these genes . In SW480 cells we also detected enhanced levels of a putative polyA HIST1HC transcript and most of the other histone genes observed by Narita and co-workers to express polyA variants following silencing of NELF or CBC (Table 1). Further, recent next generation sequencing of RNA polyadenylation has revealed the presence of polyadenylation of over twenty replication dependent histone mRNAs in unperturbed HeLa cells . In an unperturbed state the polyA histone transcripts represented ~4% of the total amount of transcription from the replication dependent histone genes. Of the over twenty histone transcripts with polyA variants detected by sequencing, we detected putative polyA versions corresponding to fifteen of these genes following CASP8AP2/FLASH LOF (Table 1). To confirm that the CASP8AP2/FLASH LOF leads to the induction or enhanced presence of the non-canonical, polyadenylated transcript variants of the replication-dependent histone genes we examined by qRT-PCR the expression of one replication-dependent histone gene HIST1H2BD. Unlike most replication-dependent histone genes where only the non-polyA transcript has been annotated as having a consensus reference sequence, two annotated transcript variants have been reported for HIST1H2BD; NM_021063, the non-polyA transcript variant and NM_138720 the polyadenylated variant (Figure 5B). Using cDNA primed with either random hexamers (Figure 5C) or oligo dT primers (Figure 5D) and primers specific for the canonical non-polyA and the poly A transcript variants (Figure 5B) we observed that following silencing of CAPS8AP2/FLASH (Figure 5Ci and 5Di) only the levels of the polyadenylated variant NM_138720 of HIST1H2BD were increased. No significant change was seen in the levels of the NM_021603/HIST1H2BD non-polyA variant (Figure 5Cii) when primed using random hexamers.
CASP8AP2/FLASH loss-of-function rapidly alters the expression of the transcriptome of SW480 cells
Complementary to our original data, NFκB transcriptional targets were once again significantly enriched within the upregulated portion of the CASP8AP2/FLASH signature at 24, 48, and 72 hours post-silencing (GSEA FDR q-values of 0.036, < 0.001, and < 0.001 respectively) (Figure 6D and 6Ei). We had anticipated that the time course data could be used to identify drivers of this transcriptional response, however, though TNF-centered knowledge based networks could be generated from the CASP8AP2/FLASH RNAi signature (Additional file 13, Figure S5) there was limited overlap between the specific genes that formed these networks and those obtained previously (Additional 8, Figure S3). We also confirmed that gene targets activated by MYC were downregulated at 24, 48, and 72 hours (all FDR q-values of < 0.001), and genes known to be suppressed by MYC were upregulated at the same time points (GSEA FDR q-values of 0.014, < 0.001, and < 0.001 at 24, 48 and 72 hours respectively) (Figure 6D and 6Eii and iii). While it is clear that transcriptional activity of MYC is reduced, we have been unable to identify upstream mediators that link these transcriptional changes to CASP8AP2/FLASH loss-of-function. Further work will thus be required to identify the specific signaling processes connecting CASP8AP2/FLASH silencing to alterations in these transcriptional networks.
All of the cyclin dependent kinase inhibitor genes identified as upregulated following CASP8AP2/FLASH silencing previously were once again upregulated, with CDKN1C (p57Kip2) once more showing the greatest change. Forty-eight hours post CASP8AP2/FLASH silencing CDKN1C/p57Kip2 mRNA levels increased ~ 4 linear fold, and this was further enhanced by 72 hours post siRNA transfection when its levels were increased by over 6 linear fold. Interestingly, one study of the expression of CDKN1C/p57Kip2 observed decreased immunostaining in colorectal carcinomas , and in our colorectal cell lines and tumor samples we saw significant reduction in expression (CDKN1C/p57Kip2 expression in CRC cell lines versus normal mucosa mean linear = 0.17 (p < 0.00098) and primary colon tumor versus normal mucosa mean linear ratio = 0.15 (p < 0.0026)). Altered expression of CDKN1C/p57/Kip2 has been noted in several cancer types, frequently as a result of epigenetic changes leading to speculation that it may act as a tumor suppressor . It will interesting to determine in the future if any of the alterations in histone gene transcription observed following the silencing of CASP8AP2/FLASH modulates the expression of epigentically regulated genes such as CDKN1C/p57Kip2. It will also be important in future studies to determine if CASP8AP2/FLASH LOF induces similar effects in other colorectal cancer cell lines and in other cancer cell types.
The silencing of CASP8AP2/FLASH enhances the expression of neurofilament heavy polypeptide (NEFH)
We have identified Caspase-8-Associated protein 2/FLICE-associated huge protein (CASP8AP2/FLASH) as essential for the survival of colorectal cancer cells. Contrary to what would perhaps be expected given its principle annotated function as a pro-apoptotic factor required for activation of the extrinsic apoptotic pathway, we observed, in the absence of FAS stimulation, significant activation of both Caspase 8 and Caspase 3/7 following inhibition of CASP8AP2/FLASH function. Instead, systems-wide analysis of the LOF of CASP8AP2/FLASH confirmed and extended biochemical studies showing that CASP8AP2/FLASH has a critical role in regulating the expression of the replication-dependent histone genes. The observation that the expression levels CASP8AP2/FLASH are the same in normal and colorectal cancer tissue also suggests a role in normal homeostasis for this protein as would be expected if its primary role is in regulating the expression of the replication-dependent histone genes. Also by using an unbiased, whole transcriptome approach we were able to identify that additional downstream effects of CASP8AP2/FLASH LOF included enhanced expression of genes targeted by NFκB and evidence of decreased MYC activity. Finally, silencing of CASP8AP2/FLASH led to expression of a recently identified tumor suppressor gene NEFH. NEFH has been shown to be the subject of epigenetic modification, as too has another gene upregulated following CASP8AP2/FLASH silencing, CDKNIC/p57. It is our current hypothesis that the changes in the expression of these genes following silencing of CASP8AP2/FLASH is likely an indirect effect; it could be that these genes are the target of one or more of the transcription factors that we see target enrichment for within the CASP8AP2/FLASH RNAi signature, or it may be that changes in the processing of the transcripts corresponding to the replication dependent histone transcripts is altering expression more broadly.
List of abbreviations
Caspase-8-Associated Protein 2
cyclin-dependent kinase inhibitor 1C (p57, Kip2)
Death-Inducing Signaling Complex
esophageal squamous cell carcinoma
FAS-associated adaptor protein
False discovery rate
FLICE-associated Huge Protein
Gene Set Enrichment Analysis
Neurofilament heavy polypetide
Normalized enrichment score
TNF receptor-associated factor 1
WD repeat domain 3
notum pectinacetylesterase homolog (Drosophila)
neurotrophic tyrosine kinase, receptor, type 1
quantitative reverse transcription PCR
small interfering RNA.
This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. ABH was supported in part by a Sallie Rosen Kaplen Fellowship from the NCI. We thank Danny Wangsa, Hesed Padilla-Nash, Chanelle Case and Sudhir Varma for technical assistance.
- Fearon ER: Molecular genetics of colorectal cancer. Annu Rev Pathol. 2011, 6: 479-507.View ArticlePubMedGoogle Scholar
- Martin SE, Caplen NJ: Applications of RNA interference in mammalian systems. Annu Rev Genomics Hum Genet. 2007, 8: 81-108.View ArticlePubMedGoogle Scholar
- Grade M, Hummon AB, Camps J, Emons G, Spitzner M, Gaedcke J, Hoermann P, Ebner R, Becker H, Difilippantonio MJ: A genomic strategy for the functional validation of colorectal cancer genes identifies potential therapeutic targets. Int J Cancer. 2011, 128: 1069-1079.PubMed CentralView ArticlePubMedGoogle Scholar
- Yang SY, Sales KM, Fuller B, Seifalian AM, Winslet MC: Apoptosis and colorectal cancer: implications for therapy. Trends Mol Med. 2009, 15: 225-233.View ArticlePubMedGoogle Scholar
- Martin SE, Jones TL, Thomas CL, Lorenzi PL, Nguyen DA, Runfola T, Gunsior M, Weinstein JN, Goldsmith PK, Lader E: Multiplexing siRNAs to compress RNAi-based screen size in human cells. Nucleic Acids Res. 2007, 35: e57-PubMed CentralView ArticlePubMedGoogle Scholar
- Camps J, Grade M, Nguyen QT, Hormann P, Becker S, Hummon AB, Rodriguez V, Chandrasekharappa S, Chen Y, Difilippantonio MJ: Chromosomal breakpoints in primary colon cancer cluster at sites of structural variants in the genome. Cancer Res. 2008, 68: 1284-1295.View ArticlePubMedGoogle Scholar
- Camps J, Nguyen QT, Padilla-Nash HM, Knutsen T, McNeil NE, Wangsa D, Hummon AB, Grade M, Ried T, Difilippantonio MJ: Integrative genomics reveals mechanisms of copy number alterations responsible for transcriptional deregulation in colorectal cancer. Genes Chromosomes Cancer. 2009, 48: 1002-1017.View ArticlePubMedGoogle Scholar
- Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J: Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 2004, 5: R80-PubMed CentralView ArticlePubMedGoogle Scholar
- Bolstad BM, Irizarry RA, Astrand M, Speed TP: A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics. 2003, 19: 185-193.View ArticlePubMedGoogle Scholar
- Smyth GK: Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol. 2004, 3: Article3-PubMedGoogle Scholar
- Hochberg Y, Benjamini Y: More powerful procedures for multiple significance testing. Stat Med. 1990, 9: 811-818.View ArticlePubMedGoogle Scholar
- Gertz EM, Sengupta K, Difilippantonio MJ, Ried T, Schaffer AA: Evaluating annotations of an Agilent expression chip suggests that many features cannot be interpreted. BMC Genomics. 2009, 10: 566-PubMed CentralView ArticlePubMedGoogle Scholar
- Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, Mesirov JP: Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA. 2005, 102: 15545-15550.PubMed CentralView ArticlePubMedGoogle Scholar
- Shen W, Wang CY, Wang XH, Fu ZX: Oncolytic adenovirus mediated Survivin knockdown by RNA interference suppresses human colorectal carcinoma growth in vitro and in vivo. J Exp Clin Cancer Res. 2009, 28: 81-PubMed CentralView ArticlePubMedGoogle Scholar
- Imai Y, Kimura T, Murakami A, Yajima N, Sakamaki K, Yonehara S: The CED-4-homologous protein FLASH is involved in Fas-mediated activation of caspase-8 during apoptosis. Nature. 1999, 398: 777-785.View ArticlePubMedGoogle Scholar
- Kim GS, Park YA, Choi YS, Choi YH, Choi HW, Jung YK, Jeong S: Suppression of receptor-mediated apoptosis by death effecter domain recruiting domain binding peptide aptamer. Biochem Biophys Res Commun. 2006, 343: 1165-1170.View ArticlePubMedGoogle Scholar
- Morgan R, Nalliah A, Morsi El-Kadi AS: FLASH, a component of the FAS-CAPSASE8 apoptotic pathway, is directly regulated by Hoxb4 in the notochord. Dev Biol. 2004, 265: 105-112.View ArticlePubMedGoogle Scholar
- Koonin EV, Aravind L, Hofmann K, Tschopp J, Dixit VM: Apoptosis. Searching for FLASH domains. Nature. 1999, 401: 662-discussion 662-663,View ArticlePubMedGoogle Scholar
- Kittler R, Putz G, Pelletier L, Poser I, Heninger AK, Drechsel D, Fischer S, Konstantinova I, Habermann B, Grabner H: An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division. Nature. 2004, 432: 1036-1040.View ArticlePubMedGoogle Scholar
- Choi YH, Kim KB, Kim HH, Hong GS, Kwon YK, Chung CW, Park YM, Shen ZJ, Kim BJ, Lee SY, Jung YK: FLASH coordinates NF-kappa B activity via TRAF2. J Biol Chem. 2001, 276: 25073-25077.View ArticlePubMedGoogle Scholar
- Jun JI, Chung CW, Lee HJ, Pyo JO, Lee KN, Kim NS, Kim YS, Yoo HS, Lee TH, Kim E, Jung YK: Role of FLASH in caspase-8-mediated activation of NF-kappaB: dominant-negative function of FLASH mutant in NF-kappaB signaling pathway. Oncogene. 2005, 24: 688-696.View ArticlePubMedGoogle Scholar
- Alm-Kristiansen AH, Saether T, Matre V, Gilfillan S, Dahle O, Gabrielsen OS: FLASH acts as a co-activator of the transcription factor c-Myb and localizes to active RNA polymerase II foci. Oncogene. 2008, 27: 4644-4656.View ArticlePubMedGoogle Scholar
- Alm-Kristiansen AH, Lorenzo PI, Molvaersmyr AK, Matre V, Ledsaak M, Saether T, Gabrielsen OS: PIAS1 interacts with FLASH and enhances its co-activation of c-Myb. Mol Cancer. 2011, 10: 21-PubMed CentralView ArticlePubMedGoogle Scholar
- Barcaroli D, Bongiorno-Borbone L, Terrinoni A, Hofmann TG, Rossi M, Knight RA, Matera AG, Melino G, De Laurenzi V: FLASH is required for histone transcription and S-phase progression. Proc Natl Acad Sci USA. 2006, 103: 14808-14812.PubMed CentralView ArticlePubMedGoogle Scholar
- Kiriyama M, Kobayashi Y, Saito M, Ishikawa F, Yonehara S: Interaction of FLASH with arsenite resistance protein 2 is involved in cell cycle progression at S phase. Mol Cell Biol. 2009, 29: 4729-4741.PubMed CentralView ArticlePubMedGoogle Scholar
- Bongiorno-Borbone L, De Cola A, Barcaroli D, Knight RA, Di Ilio C, Melino G, De Laurenzi V: FLASH degradation in response to UV-C results in histone locus bodies disruption and cell-cycle arrest. Oncogene. 2010, 29: 802-810.View ArticlePubMedGoogle Scholar
- Barcaroli D, Dinsdale D, Neale MH, Bongiorno-Borbone L, Ranalli M, Munarriz E, Sayan AE, McWilliam JM, Smith TM, Fava E: FLASH is an essential component of Cajal bodies. Proc Natl Acad Sci USA. 2006, 103: 14802-14807.PubMed CentralView ArticlePubMedGoogle Scholar
- Bongiorno-Borbone L, De Cola A, Vernole P, Finos L, Barcaroli D, Knight RA, Melino G, De Laurenzi V: FLASH and NPAT positive but not Coilin positive Cajal Bodies correlate with cell ploidy. Cell Cycle. 2008, 7: 2357-2367.View ArticlePubMedGoogle Scholar
- Yang XC, Burch BD, Yan Y, Marzluff WF, Dominski Z: FLASH, a proapoptotic protein involved in activation of caspase-8, is essential for 3' end processing of histone pre-mRNAs. Mol Cell. 2009, 36: 267-278.PubMed CentralView ArticlePubMedGoogle Scholar
- Yang XC, Xu B, Sabath I, Kunduru L, Burch BD, Marzluff WF, Dominski Z: FLASH is required for the endonucleolytic cleavage of histone pre-mRNAs but is dispensable for the 5' exonucleolytic degradation of the downstream cleavage product. Mol Cell Biol. 2011,Google Scholar
- Burch BD, Godfrey AC, Gasdaska PY, Salzler HR, Duronio RJ, Marzluff WF, Dominski Z: Interaction between FLASH and Lsm11 is essential for histone pre-mRNA processing in vivo in Drosophila. RNA. 2011, 17: 1132-1147.PubMed CentralView ArticlePubMedGoogle Scholar
- Dominski Z: An RNA end tied to the cell cycle: new ties to apoptosis and microRNA formation?. Cell Cycle. 2010, 9: 1308-1312.View ArticlePubMedGoogle Scholar
- Jeong EG, Lee SH, Lee HW, Soung YH, Yoo NJ: Immunohistochemical and mutational analysis of FLASH in gastric carcinomas. APMIS. 2007, 115: 900-905.View ArticlePubMedGoogle Scholar
- Milovic-Holm K, Krieghoff E, Jensen K, Will H, Hofmann TG: FLASH links the CD95 signaling pathway to the cell nucleus and nuclear bodies. EMBO J. 2007, 26: 391-401.PubMed CentralView ArticlePubMedGoogle Scholar
- Degenhardt Y, Lampkin T: Targeting Polo-like kinase in cancer therapy. Clin Cancer Res. 2010, 16: 384-389.View ArticlePubMedGoogle Scholar
- Eilers M, Eisenman RN: Myc's broad reach. Genes Dev. 2008, 22: 2755-2766.PubMed CentralView ArticlePubMedGoogle Scholar
- Vervoorts J, Luscher-Firzlaff J, Luscher B: The ins and outs of MYC regulation by posttranslational mechanisms. J Biol Chem. 2006, 281: 34725-34729.View ArticlePubMedGoogle Scholar
- Marzluff WF, Gongidi P, Woods KR, Jin J, Maltais LJ: The human and mouse replication-dependent histone genes. Genomics. 2002, 80: 487-498.View ArticlePubMedGoogle Scholar
- Mannironi C, Bonner WM, Hatch CL: H2A.X. a histone isoprotein with a conserved C-terminal sequence, is encoded by a novel mRNA with both DNA replication type and polyA 3' processing signals. Nucleic Acids Res. 1989, 17: 9113-9126.PubMed CentralView ArticlePubMedGoogle Scholar
- Marzluff WF, Wagner EJ, Duronio RJ: Metabolism and regulation of canonical histone mRNAs: life without a poly(A) tail. Nat Rev Genet. 2008, 9: 843-854.PubMed CentralView ArticlePubMedGoogle Scholar
- Narita T, Yung TM, Yamamoto J, Tsuboi Y, Tanabe H, Tanaka K, Yamaguchi Y, Handa H: NELF interacts with CBC and participates in 3' end processing of replication-dependent histone mRNAs. Mol Cell. 2007, 26: 349-365.View ArticlePubMedGoogle Scholar
- Shepard PJ, Choi EA, Lu J, Flanagan LA, Hertel KJ, Shi Y: Complex and dynamic landscape of RNA polyadenylation revealed by PAS-Seq. RNA.
- Li JQ, Wu F, Usuki H, Kubo A, Masaki T, Fujita J, Bandoh S, Saoo K, Takeuchi H, Kuriyama S: Loss of p57KIP2 is associated with colorectal carcinogenesis. Int J Oncol. 2003, 23: 1537-1543.PubMedGoogle Scholar
- Pateras IS, Apostolopoulou K, Niforou K, Kotsinas A, Gorgoulis VG: p57KIP2: "Kip"ing the cell under control. Mol Cancer Res. 2009, 7: 1902-1919.View ArticlePubMedGoogle Scholar
- Kim MS, Chang X, LeBron C, Nagpal JK, Lee J, Huang Y, Yamashita K, Trink B, Ratovitski EA, Sidransky D: Neurofilament heavy polypeptide regulates the Akt-beta-catenin pathway in human esophageal squamous cell carcinoma. PLoS One. 2010, 5: e9003-PubMed CentralView ArticlePubMedGoogle Scholar
- Torisu Y, Watanabe A, Nonaka A, Midorikawa Y, Makuuchi M, Shimamura T, Sugimura H, Niida A, Akiyama T, Iwanari H: Human homolog of NOTUM, overexpressed in hepatocellular carcinoma, is regulated transcriptionally by beta-catenin/TCF. Cancer Sci. 2008, 99: 1139-1146.View ArticlePubMedGoogle Scholar
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