Genes that are highly overexpressed in tumor cells can be required for tumor cell survival and have the potential to be selective therapeutic targets. In an attempt to identify such targets, we combined a functional genomics and a systems biology approach to assess the consequences of RNAi-mediated silencing of overexpressed genes that were selected from 140 gene expression profiles from colorectal cancers (CRCs) and matched normal mucosa. In order to identify credible models for in-depth functional analysis, we first confirmed the overexpression of these genes in 25 different CRC cell lines. We then identified five candidate genes that profoundly reduced the viability of CRC cell lines when silenced with either siRNAs or short-hairpin RNAs (shRNAs), i.e., HMGA1, TACSTD2, RRM2, RPS2 and NOL5A. These genes were further studied by systematic analysis of comprehensive gene expression profiles generated following siRNA-mediated silencing. Exploration of these RNAi-specific gene expression signatures allowed the identification of the functional space in which the five genes operate and showed enrichment for cancer-specific signaling pathways, some known to be involved in CRC. By comparing the expression of the RNAi signature genes with their respective expression levels in an independent set of primary rectal carcinomas, we could recapitulate these defined RNAi signatures, therefore, establishing the biological relevance of our observations. This strategy identified the signaling pathways that are affected by the prominent oncogenes HMGA1 and TACSTD2, established a yet unknown link between RRM2 and PLK1 and identified RPS2 and NOL5A as promising potential therapeutic targets in CRC.
The application of parallel gene expression profiling techniques to large sets of primary tumor samples has revealed profound alterations in the cancer transcriptome. The catalogs of deregulated genes defined by such approaches are not only important because they provide new insight into tumor biology but also because they reveal those genes that are specifically upregulated in tumors, some of which may represent promising new antitumor molecular targets.
Finding such critical genes within a complex expression signature, however, remains a formidable challenge. One possible approach to this problem is to modulate the expression of these genes in cell line models. For example, loss-of-function (LOF) analysis can be used to identify genes whose reduction of expression may have a direct impact on cancer cell survival.
We recently presented comprehensive gene expression signatures of colorectal cancer (CRC) and matched normal mucosa.1, 2 In order to identify those genes that could directly influence tumor survival and thus, may represent candidate molecular targets, we first identified CRC cell lines in which the expression of these genes was upregulated accordingly. We then used RNAi-mediated gene silencing to reduce their expression and quantified cell survival. To uncover their roles in cell viability, we monitored genome-wide transcriptional consequences of silencing these candidate genes. This allowed the identification of the cellular interaction networks, and, by comparing these signatures with gene expression profiles of independent primary tumors, established the clinical relevance of our approach.
Material and Methods
Cell culture, RNA isolation and real-time PCR
Twenty-four CRC cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA): SW480, HT-29, SW837, SW48, T84, LS 513, HCT 116, SW1463, SW1116, LS 411N, NCI-H716, LoVo, NCI-H508, COLO 201, COLO 320DM, LS 123, LS 174T, DLD-1, Caco-2, LS 1034, SK-CO-1, RKO, SW403 and SW620. p53HCT 116 cells were a gift from Bert Vogelstein. All cell lines were cultured in the recommended media containing 10% fetal bovine serum, and cell line cross-contamination was excluded by short tandem repeat profiling.3 Total RNA was isolated using TRIzol (Invitrogen, Gaithersburg, MD) and purified using the RNeasy Mini kit (Qiagen, Germantown, MD). Detailed protocols are at http://www.riedlab.nci.nih.gov/. Nucleic acid quantity, quality and purity were determined using a spectrophotometer (Nanodrop, Rockland, DE) and a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Subsequently, real-time PCR was performed as previously described.2 Details are available in Supporting Information Methods.
The target sequences for synthetic siRNA duplexes (Qiagen, Germantown, MD) are listed in Supporting Information Table 1. Lipid-based reverse transfections were performed as previously described.4, 5 Individual Expression Arrest™ lentiviral inducible short-hairpin RNA constructs (Open Biosystems, Huntsville, AL) were transfected using Nucleofector technology (Amaxa, Cologne, Germany), and stable single-cell clones were subsequently expanded. For siRNAs, gene-specific RNAi was measured at an mRNA level using either the QuantiGene Reagent System as previously described5 or a custom QuantiGene Plex 1.0 Reagent System (Panomics, Fremont, CA; Supporting Information Table 2). Short-hairpin RNA (shRNA) knockdown was validated using real-time PCR. Details are available in Supporting Information Methods.
Cell viability assay
Cell viability was determined using the CellTiter-Blue® reagent (Promega, Madison, WI). Replicate transfections per siRNA duplex were set up for every time point, and reduction of resazurin to resorufin was measured 72 hr (data not shown) and 96 hr after transfection using a plate reader (Spectramax 2e, Molecular Devices, Sunnyvale, CA). Viability of transfected cells was compared with cells transfected with a negative control siRNA (Qiagen). Similarly, expression of shRNAmir clones was induced as described above, and viability was determined 72 hr (data not shown) and 96 hr after induction compared with cells expressing a negative control shRNAmir (Open Biosystems).
Gene expression profiling following RNAi
Triplicate transfections were independently performed with two different siRNA duplexes corresponding to each gene target. As a negative control, we used a siRNA duplex (AllStar siNegative; Qiagen) that has been experimentally validated as generating minimal non-specific effects on global gene expression levels and cellular phenotypes. Total RNA was isolated from SW480 cells transfected with siRNAs corresponding to HMGA1, RRM2 and RPS2 48 hr post-transfection and 72 hr post-transfection for siRNAs corresponding to TACSTD2 and NOL5A. Different time points were chosen because maximal reduction of viability upon inhibition of HMGA1, RRM2 and RPS2 was already observed after 72 hr, whereas inhibition of TACSTD2 and NOL5A reached maximal levels after 96 hr. Subsequently, a reduction in target mRNA levels was confirmed by real-time PCR (Supporting Information Table 1), and gene expression profiling was performed as previously described.6 The respective gene expression data have been deposited in the NCBI Gene Expression Omnibus (GSE15212).
Statistical analysis of microarray data
Array data were log2 transformed, normalized and corrected for multiple testing. Gene lists for the different comparisons (each experimental siRNA separately vs. negative-control siRNA) were generated based on stringent cutoff criteria (q-value of <0.05 and linear fold change of >∼1.5). Because two different siRNAs were used to target the same gene, the overlap of these lists was generated using the additional criterion that the regulation should point in the same direction. A detailed description is available in Supporting Information Methods.
Estimation of signal flow networks and pathway analysis
Signal flow networks from RNAi effects in gene expression were reconstructed with our nested effects models approach7 using those genes that were selected according to the conditions described above. To further determine the functional relationships between genes within the RNAi signatures, Ingenuity Pathway Analysis tools (Ingenuity Systems, Redwood City, CA) were used. Details are available in Supporting Information Methods.
Comparison of cell line and tumor gene expression profiles via enrichment analysis
To compare these RNAi signatures with gene expression profiles recently generated from an independent set of 65 primary rectal cancers and matched normal mucosa samples (Gaedcke et al., in preparation), we computed lists of differentially expressed genes (cutoff criterion of FDR <0.05). We then determined the overlap of these lists and the sets of probes deregulated after silencing of a given candidate gene, and we evaluated the significance of an overrepresentation using a one-sided Fisher's exact test. Further details are available in Supporting Information Methods.
Protein expression levels of RPS2 in CRCs and normal mucosa were assessed on tissue microarrays. Technical details are available in Supporting Information Methods.
CRC cell lines recapitulate the gene expression pattern of primary tumors
The general outline of our experimental strategy is presented in Figure 1a. We recently established gene expression signatures of primary CRCs and identified many genes that were highly overexpressed in the tumors.1, 2 In order to identify model systems to study the relevance of such overexpressed genes for cell viability and hence as potential therapeutic targets, we first measured the expression levels of 25 highly upregulated genes in 25 CRC cell lines using real-time PCR (Supporting Information Table 3). Eleven of these 25 genes were highly overexpressed in the majority of tumor cell lines and were, therefore, carried forward for functional validation (Fig. 1b and Table 1). Together with the maintenance of specific patterns of genomic imbalances, the preservation of gene expression changes in CRC cell lines is consistent with data from breast cancer cell lines8 and from other tumor models, which suggest that they are credible models for meaningful functional analysis.9, 10
Table 1. List of 11 genes that were consistently overexpressed in primary CRC, and in CRC cell lines, respectively
Loss-of-function analysis identifies genes required for cell viability
The 11 genes selected for LOF analysis using RNAi were studied in SW480 using two synthetic siRNA duplexes per gene. Transfection of 15 of 22 siRNAs (68%) decreased mRNA levels of the corresponding target gene by >70% relative to a control siRNA (siNeg) 48 hr post-transfection (Supporting Information Table 1). Silencing of seven genes, HMGA1, MYC, NOL5A, TACSTD2, RAN, RPS2 and RRM2, mediated a 20% or greater decrease in cellular viability (Supporting Information Fig. 1). Obviously, we were reassured to identify MYC because it validates our general experimental approach. MYC is frequently amplified, overexpressed and activated via chromosomal translocations in human cancer.11 However, due to the wealth of knowledge on MYC signaling, we did not include MYC in further experimentation. Accordingly, the five final candidate genes were TACSTD2, HMGA1, NOL5A, RPS2 and RRM2 (Fig. 2a).
siRNA duplexes have the potential to interact with other transcripts as a result of microRNA-like interactions.12, 13 One approach to reducing the probability that an observed phenotype is due to non-specific effects is to show that additional siRNA duplexes with different sequences lead to similar phenotypic consequences. Following this approach, we observed reduction of viability with at least three siRNAs (Supporting Information Fig. 1).
Because the RNAi machinery can also be harnessed to suppress gene expression using shRNAs, we wished to reproduce our siRNA results employing shRNA expression cassettes (with different sequences from that of the siRNAs). We therefore established stable SW480 clones that can be induced to express shRNAmirs-targeting RRM2, RPS2 and NOL5A (the genes for which shRNAs were available). In all instances, the shRNA-mediated decreased cell viability was very similar to that mediated by synthetic siRNAs (Fig. 2b).
To ensure that the effect on cell viability is not limited to SW480, we transfected the CRC cell lines HT-29 and DLD-1, for which we had confirmed overexpression of the candidate genes. Silencing of NOL5A, RPS2 and RRM2 reduced the viability of both cell lines (Fig. 2c).
Generation of loss-of-function-specific transcriptional signatures
To explore the cellular pathways in which the candidate genes operate, we conducted whole transcriptome expression analyses of siRNA-transfected SW480 cells to identify an “RNAi signature” for each gene of interest. These RNAi signatures were defined as the overlap of genes with altered expression following transfection of SW480 with two different siRNA duplexes compared with a negative control. We then systematically explored whether any of the gene expression changes that we observed could be because of non-sequence-specific or sequence-specific microRNA-like “off-target” interactions,14–16 and removed genes with potential interactions from our lists (details are available in Supporting Information Results). Based on these criteria, the number of deregulated probes amounted to 324 for RRM2, 187 for NOL5A, 136 for RPS2, 123 for HMGA1 and 76 for TACSTD2 (Supporting Information Table 4). In all instances, the target gene corresponding to each siRNA was successfully silenced as evidenced by a profound reduction in target transcript levels measured by both real-time PCR (Supporting Information Table 1) and expression microarray (Supporting Information Table 4). The cluster analysis (Fig. 3a) visualizes the results and illustrates experimental reproducibility. Furthermore, the remarkably high correlation in the changes in gene expression that we observed following RNAi with two different siRNA duplexes suggests that the vast majority of these modulations are a functional consequence of gene-specific silencing and not because of non-specific effects (Fig. 3b).
Prediction of functional relationships from gene-specific RNAi signatures
Although the established or proposed functions of RPS2, NOL5A, RRM2, HMGA1 and TACSTD2 are diverse, we were interested to see if the respective RNAi signatures contained common transcriptional features that indicate functional relationships between these overexpressed CRC genes, and, thus, reflect critical processes altered in CRC. To assess this, we used nested effects models17, 18 to estimate signal-flow network relationships. The principal idea of this approach is that the silencing of a gene that is high in a signaling cascade will influence the expression of many downstream genes, whereas the silencing of a gene that acts lower in the cascade will generate effects in only a subset of these genes. Seven hundred forty unique probes were selected for analysis (Supporting Information Table 4). The results are shown in the left part of Figure 4, and the genes that contributed to the NEM are listed in Supporting Information Table 5. This analysis suggests that TACSTD2 is below the other genes because its signature has the most overlap with the other RNAi signatures. The RNAi signatures for HMGA1, RRM2 and RPS2 suggest these influence separate processes as each display substantial independent effects. The NOL5A RNAi signature overlaps with the RRM2 RNAi signature but many of these changes are not seen within the TACSTD2 RNAi signature. The nested effect models predicted relationships for the CRC-overexpressed genes are displayed on the right part of Figure 4. The placement of HMGA1, RRM2 and RPS2 high in the hierarchy of this model is consistent with the observation that these genes were associated with the most pronounced reduction of cell viability (Fig. 2a).
Biological networks perturbed by gene-specific loss-of-function
Silencing of NOL5A, RPS2, HMGA1, TACSTD2 and RRM2 in CRC cell lines induced a substantial reduction of cell viability although with different kinetics and to a different degree. The broad mechanism for the effect on cell survival following LOF is firmly established for RRM2, as it is required for DNA synthesis,19 and for HMGA1, which acts as transcription factor and oncogene.20 However, it is less well defined or unknown for the other genes. We, therefore, used computational pathway analysis tools to interrogate the LOF-RNAi signatures for each gene to identify critical biological networks and to identify commonalities. It should be noted that, although our phenotypic endpoint was reduction of cell viability, we saw minimal changes in the expression of genes involved in apoptotic or other cell death-related pathways. This suggests that we have captured a picture of change in gene expression before cell death.
The RNAi signature for RRM2 stood out from the other signatures because almost 90% of the deregulated genes were upregulated as a consequence of RRM2 silencing. The RRM2 RNAi signature genes were connected through extracellular signal-regulated kinase (ERK) and three members of the cyclin family (Fig. 5a) or through CTNNB1 (Fig. 5b). Additional networks centered on HNF4A, TGFB1, MAPK14 and NFkB, TNF and STAT3, SMARCA4, ERBB2, SMAD2 and TP53 (Supporting Information Fig. 2).
Within the TACSTD2 signature, the next most downregulated gene after the target itself was RRM2 (Supporting Information Fig. 3). Networks that included TACSTD2 itself showed HIF1A and NHF4A as connecting focus genes (Supporting Information Fig. 3). Two other networks reflect TP53 signaling and the ras-PI3k-Akt axis (Supporting Information Fig. 3).
We identified five networks associated with the HMGA1 RNAi signature: in the first, the focus genes are connected through TP53, the second was enriched in insulin-like growth factor (IGF) signaling and the third was defined by the ras-PI3k-Akt axis (Supporting Information Fig. 4). In these three networks, HMGA1 was included as a downregulated focus gene. In addition to that, we identified two networks that were connected solely through genes that were deregulated as a consequence of HMGA1 LOF, i.e., HMGA1 did not appea as a focus gene (Supporting Information Fig. 4). One of these networks was connected via the focus genes NFkB and STAT3, the second showed HNF4A as the central node. These two networks, therefore, describe yet unknown HMGA1-associated pathways (Supporting Information Fig. 4).
Interestingly, many of the transcripts within the RPS2 RNAi signature have minimal annotation and/or functional data. However, pathway analysis showed major nodes of network connections centered around MAPK1 in one network (Supporting Information Fig. 5), HNF4A in another one (Fig. 5c) and TGFB1 in a third network (Supporting Information Fig. 5). This third network was also enriched for multiple members of the matrix metalloproteinase gene family ADAM. The network in which RPS2 was downregulated as a focus gene was connected through CCNE1, ERK and the translation initiation factor EIF4EBP1 (Fig. 5d).
When studying the deregulated genes associated with NOL5A silencing, we identified a network connected through ERK, MAPK14, CCNE1 and SERPINE1 (Fig. 5e). As observed for other target genes as well, another network was connected through HNF4A (Fig. 5f). Other networks included TGFB1, NFkB and SMARCA4, CTNNB1 or TP53 (Supporting Information Fig. 6). The latter was the only network that included the downregulated NOL5A as focus gene. Interestingly, we observed that another network exhibited many focus genes as central hubs including JUN, FOS, SP1, HIF1A, VEGFA and ERBB2 (Supporting Information Fig. 6).
Although most of the transcriptional changes within each RNAi signature were specific for each silenced gene, there was an interesting overlap in the biological networks (Supporting Information Table 6), consistent with the nested effect models analysis. For instance, for many RNAi signatures, we generated networks with connecting nodes that included the focus genes TP53, CTNNB1 (β-catenin), NFKB, TGFB and ERK, all of which are relevant for colorectal carcinogenesis. In addition, HNF4A played a dominant connecting role for all five genes, confirming recent findings demonstrating that inhibition of HNF4A activity inhibits colorectal carcinoma cell line growth in vitro and in vivo.21
Comparison of RNAi signatures with expression profiles of primary rectal cancer
If overexpression of a gene in primary CRC suppresses the transcription of a network of genes, we speculated that silencing of this gene should lead to the upregulation of at least some of these genes within the RNAi signature. Conversely, if overexpression of a gene in primary CRC activates the transcription of a network of genes, then silencing of this gene should lead to the downregulation of these genes within the RNAi signature (Supporting Information Fig. 7). We tested this hypothesis by performing an enrichment analysis (see Material and Methods). Supporting Information Figure 7 shows a statistically significant inverse correlation for NOL5A, RRM2 and RPS2 RNAi signatures vs. tumor profiles, i.e., upregulation of signature genes that are downregulated in primary carcinomas in cell lines after RNAi, and vice versa, validating our approach by lending weight to their biological significance.
Robust array technologies allow global surveillance of tumor transcriptomes and matched normal tissues. Differentially expressed genes are likely to be involved in tumorigenesis and can, therefore, serve as potential molecular targets for rational cancer therapy. However, the interpretation of global expression signatures in terms of their functional relevance remains a major challenge.
We, therefore, explored a LOF approach to systematically investigate the role of overexpressed genes derived from previously established gene expression signatures of primary CRCs for viability of CRC cells.1, 2 Our experimental strategy led us to focus on HMGA1, RRM2, TACSTD2, NOL5A and RPS2 as important candidate genes because their downregulation diminishes the viability of CRC cell lines. Systematic mRNA expression analyses following RNAi-mediated gene silencing unveiled unique RNAi expression signatures pointing to critical transcriptional responses. The underlying deregulation of cellular pathways within these RNAi signatures could be recapitulated in an independent data set of 65 rectal carcinomas. To the best of our knowledge, we are the first to systematically combine gene expression profiling of primary tumors and extensive expression profiling after RNAi-mediated gene silencing. This approach proved exceedingly robust.
Although the independent identification of RRM2, TACSTD2 and HMGA1 provides elegant proof-of-concept for our experimental strategy, the suggested role of NOL5A and RPS2 as potential therapeutic targets is a novel finding. RRM2 confers the enzymatic activity of the ribonucleotide reductase complex, which represents the rate-limiting enzyme in DNA synthesis and as such is required for DNA replication and DNA repair.19 It was previously shown that RRM2 is upregulated in CRCs22 and that its inhibition reduced the proliferation of cancer cells, triggering the development of RRM2-targeted siRNA-containing nanoparticles.23, 24 Our observations confirm that this gene has enormous potential as a therapeutic target. Importantly, we are the first to report a yet undiscovered link between RRM2 and PLK1, which was one of the most drastically downregulated genes following RRM2 silencing (Supporting Information Table 4 and Fig. 5A). PLK1 encodes the Polo-like kinase 1 protein, a key regulator of cell division. Inhibition or depletion of PLK1 is associated with activation of apoptosis and it is actively being pursued as an anticancer target.25 Because both proteins are in the focus of current anticancer therapeutic development efforts, this may open a new entry-point for combinatorial treatment.
Our data for TACSTD2 are consistent with the emerging role for this cell-surface receptor in CRC and potentially other cancers.26, 27 Recently, Wang et al.28 demonstrated that expression of TACSTD2 was required for the tumorigenic and invasive potential of CRC cells, and TACSTD2 has been proposed as a marker for prostate basal cells with stem cell characteristics.29 Of note, a therapeutic antibody targeting TACSTD2 was described recently.30 Our data would warrant exploring the use of this antibody in CRC. Interestingly, we identified RRM2 as one of the highly downregulated genes within the TACSTD2 RNAi signature, suggesting a potential functional link.
HMGA1 is a known transcription factor and colorectal oncogene.31 However, despite the wealth of information on this promising target, there are still no specific compounds in preclinical development. Itself a target of c-Jun, caseine kinase 2, protein kinase, Ubc9, the ataxia telangiectasia mutated (ATM) kinase and—most recently—miR-16, it has been described from biochemical studies as a modulator of the Ras/ERK mitogenic signaling pathway, including KIT ligand and caveolins 1 and 2, and consequently a negative regulator of mechanistic target of rapamycin (mTOR) phosphorylation (additional pertinent references can be found in Supporting Information References). Because this is the first report elucidating the transcriptional pathways modulated by HMGA1, we anticipate that this might help to identify downstream targets that affect the same pathway and that can be inhibited by more traditional means.
The NOL5A gene encodes the nucleolar protein 5A, which is thought to be involved in the assembly of the 60S ribosomal subunit.32 Little is known about this gene or the druggable characteristics of its protein product, although several intriguing observations have recently been made. As a component of the ribonucleoprotein methylation complex, it is known to colocalize with the putative methyl transferase fibrillarin and appears to associate with the nuclear phosphoprotein treacle specifically in telophase, but not during mitosis. It is also one of several genes whose downregulation was correlated with acquired chemotherapy resistance in vivo and in multicellular tumor spheroids, but not in monolayers (additional pertinent references can be found in Supporting Information References). To the best of our knowledge, NOL5A has not previously been linked to human malignancies.
The ribosomal protein S2, RPS2, comprises a component of the 40S ribosomal subunit. Its exact function remains to be determined, although RPS2 has been identified as a substrate of the ribosomal protein methyltransferase, protein arginine methyltransferase 3.33 Recently, elevated mRNA levels have been associated with increased cellular proliferation, and overexpression of RPS2 has been detected in liver tumors, astrocytomas and prostate cancers.34–36 Additionally, RPS2 has been identified as a novel tyrosine kinase substrate in breast cancer.37 Its important role for the viability of CRC cells is a novel finding, which is substantiated by the fact that the RPS2 protein is not expressed in colon mucosa, but in the majority (72%) of tumor samples based on immunohistochemical staining (Supporting Information Fig. 8).
Interestingly, NOL5A and RPS2 both carry the conserved E-box canonical MYC-binding sequence in their promoter region. MYC has been considered for many years as an attractive therapeutic target. However, its activity has been hard to inhibit specifically,38 and targeting critical downstream proteins may be a more realistic approach. Importantly, we observed that the expression levels of MYC and both RPS2 and NOL5A are positively correlated in 65 primary rectal carcinomas (Supporting Information Fig. 9). Although there is no formal proof yet that MYC actually binds to regulatory elements of these two genes in CRC, our analysis provides the first experimental evidence that they might be functionally linked. Furthermore, the expression levels of MYC and RPS2 show a positive correlation in lymphomas,39 and it has been reported that the expression levels of NOL5A increase in the presence of MYC.40–42
Taken together, these results demonstrate the power of our strategy combining a functional genomics and a systems biology approach. We confirmed the involvement of HMGA1, RRM2 and TACSTD2 and defined their signaling pathways in the context of CRC, which may now enable identification of alternative means for targeting these genes. In addition, our RNAi signatures revealed other attractive candidates that have not yet been considered as target genes in CRC. Among the genes commonly deregulated after gene silencing, we identified the plausible upregulation of ANGPTL4, BRMS1L and RND3 (RhoE). ANGPTL4 has been previously shown to block tumor cell invasiveness and metastases,43BRMS1L represents a metastasis suppressor gene44 and RND3 inhibits Ras-induced cellular transformation.45 We also observed the downregulation of HAS2 and ID2, overexpression of which triggers tumorigenesis and progression of breast cancer cells,46 and promotes survival of CRC cells.47 Most importantly, we identified NOL5A and RPS2 as potential novel therapeutic targets.
The authors are indebted to Ms. Jessica Eggert, Ms. Antje Schneeberg, Mr. Chan Rong Lai and Ms. Tamara Jones for excellent technical support. We also thank Drs Daniel Soppet and Scott E. Martin for helpful discussions, Dr. Gabriela Salinas-Riester and Mr. Lennart Opitz for their help with microarray hybridizations, Dr. Michael Klintschar for short tandem repeat profiling analysis and Mr. Buddy Chen for editorial assistance.