Systems Analysis of the NCI-60 Cancer Cell Lines By Alignment of Protein Pathway Activation Modules with "-OMIC" Data Fields and Therapeutic Response Signatures

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Introduction
The NCI-60 cell lines are a set of human tumor cell lines collected by the National Cancer Institute (NCI) over the last 20 years to accomplish drug screening tests of more than 100,000 chemical compounds and natural extracts (1).This panel represents the most common solid and soft tumors derived from 9 different tissues such as blood, lung, colon, kidney, breast, skin, prostate, ovary, and central nervous system.The NCI-60 cell line set is used in labo-ratories throughout the world as in vitro tumor models, thanks to their advantages of reproducibility, availability, and representation of tumor site lineages.Most recently, the NCI-60 cell lines have been characterized by a number of high-throughput molecular profiling efforts through DNA mutations (2), RNA (3), single-nucleotide polymorphisms (4), miRNAs (5), metabolomic, proteomic (6), and karyotyping (7) screens, which have led to a better understanding of the biology of these cell lines, and an increased understanding concerning the relationships between therapeutic resistance/sensitivity and the underpinning biology.Many scientists are now focusing on protein-based analysis for these types of studies because of the proximity of the proteome to the mechanism of action of most therapeutics and their primary relationship to cellular biochemistry.Past efforts used techniques such as 2-dimensional PAGE, mass spectrometry-based profiling, and both antibody and protein microarrays (8).
Reverse-phase protein microarray (RPPA) technology (9), in particular, has been used to gain better insights into the expression profiles of the NCI-60 set due to its ability to quantitatively measure a large number of protein analytes at once with extremely high sensitivity, and was used to measure a subscribed set of proteins and phosphoproteins from the NCI-60 cell lines (6,10).However, these past efforts have failed to systematically interrogate the signaling architecture of this important set using broad-scale, functional phosphoproteomic mapping within the context of a focused network-oriented approach.In our work described herein, we used RPPA to measure the activation/phosphorylation state of 135 key signaling proteins, and a total of nearly 200 protein endpoints (i.e., cleaved, phosphorylated, and/or total protein isoforms) involved in many aspects of tumorigenesis and metastatic progression and representing drug targets for a number of current and experimental therapeutics across the entire 60 lines that comprise the NCI-60 set.We aggregated these individual protein measurements in a system-based, pathway-oriented manner that was then used to interrogate these pathway activity profiles more deeply within the context of the myriad of "-omic" measurements and coordinate enormous existing drug sensitivity data sets.This analysis uncovered new biochemical linkages between drug sensitivities, RNA, DNA, protein, phosphoprotein, and metabolomic profiles, and could provide a beginning step to fully link drug sensitivity and "-omic"-based interconnections to a network-focused view of tumor biology.

Reverse-phase protein array analysis
RPPAs were constructed and analyzed as previously described (9).Briefly, samples from the replicate sets of the 60 cell lines were printed in triplicate spots on nitrocellulosecoated glass slides (GRACE Bio-Labs) using an Aushon 2470 arrayer equipped with 185 mm pins (Aushon Biosystems), according to the manufacturer's instructions.Reference standard lysates, composed of HeLa þ Pervanadate (BD), Jurkat þ Etoposide (Cell Signaling), and Jurkat þ Calyculin A (Cell Signaling) cell lysates, were printed in 10point dilution curves as procedural controls and as positive controls for antibody staining.Each reference standard curve was printed in triplicate at concentrations of 0.5 mg/mL and 0.125 mg/mL.A selected subset of the printed array slides were stained with Sypro Ruby Protein Blot Stain (Invitrogen) to estimate sample total protein concentration, and the remaining slides were stored desiccated at À20 C. Just before antibody staining, printed slides were treated with 1Â ReBlot Mild Solution (Chemicon) for 15 minutes, washed 2 times for 5 minutes with PBS (Invitrogen), and incubated for 1 hour in blocking solution [2% I-Block (Applied Biosystems), 0.1% Tween-20 in PBS].Immunostaining was completed on an automated slide stainer using a catalyzed signal amplification kit (DAKO).The arrays were probed with a library of almost 200 antibodies against total, cleaved, and phosphoprotein endpoints (Supplementary Table S1).Primary antibody binding was detected using a biotinylated goat anti-rabbit immunoglobulin G (IgG) HþL (1:7500; Vector Laboratories) or rabbit anti-mouse IgG (1:10; DAKO) followed by streptavidin-conjugated IRDye680 fluorophore (LI-COR Biosciences).Before use, primary antibodies were extensively validated for single-band specificity by Western immunoblotting with complex cellular lysates.Negative control slides were incubated with secondary antibody only.All Sypro and immunostained slides were scanned using a Revolution 4550 scanner (Vidar Corp.), and acquired images were analyzed with MicroVigene v4.0.0.0 (VigeneTech), which conducted spot detection, local background subtraction, negative control subtraction, replicate averaging, and total protein normalization, producing a single value for each sample.Unsupervised hierarchical clustering was conducted with JMP v5.1 (SAS Institute).Endpoint relative intensity correlation plots were conducted with GraphPad Prism v5 (GraphPad Software Inc.,).

Protein pathway activation score determination
Protein signaling pathway activation modules for AKT, mTOR, EGF receptor (EGFR), insulin-like growth factor-1 receptor (IGF-1R), integrin, and apoptosis signaling were defined on the basis of known biochemical linkages between the individual phosphoproteins quantitatively measured by RPPA.Pathway activation module scores were calculated by first scaling the relative intensity values within each endpoint to the sample with the highest value, resulting in values ranging from 1 to 0 that were designated as the "single endpoint score".Second, final pathway activation module scores for each sample were generated by summing the single individual phosphoprotein score for each endpoint component in a given module.These scores, referred to as an "overall module score" or "pathway activation score," represent the whole activation status of each of the 6 pathways under consideration for each cell line.

"-Omic" network analysis
We downloaded the following normalized datasets for the NCI-60 cell lines from CellMiner (11): mRNA expression measured using Affymetrix HG-U133, miRNA expression measured using Agilent Human miRNA microarray and OSU V3 chip, DNA copy number measured with Onco-BAC DNA microarrays, and DNA mutation data.From the same source, we also downloaded drug responses measured as Àlog(GI 50 ) for datasets A118.From NCI DTP (12), we downloaded the Metabolon metabolomic dataset, Sequenom DNA methylation dataset, and drug responses for 97 U.S. Food and Drug Administration-approved anticancer drugs.
From the mRNA expression data, we inferred coexpression networks using ARACNe (13), CLR (14), MIRNET (15), C3NET (16), and GENIE3 (17).In the first 3 methods, we constructed 2 networks for each method, using Spearman rank coefficient and mutual information as a relationship measure.From the 8 resulting gene coexpression networks, we constructed a single consensus network, by taking edges that were present in all 8 networks, and also edges that were present in at least 6 networks if the 2 genes for the edge were significantly positively or negatively correlated at 2-tailed P 0.05 after Bonferroni correction.We repeated the same procedure with phosphoprotein data, arriving with a consensus protein coexpression network.
From the inferred cell-wide protein and gene consensus coexpression networks, we narrowed our focus to networks centered around individual pathway activation scores.Each such network consists of proteins, genes, and other entities that were directly correlated with the score.We calculated Pearson correlation coefficients, with Bonferroni-adjusted 2tailed P ¼ 0.05 cutoff, to detect genes (mRNA expression), DNA mutation, methylation and copy number, metabolites, and drug sensitivities that are directly correlated with each pathway activation score.For DNA mutation, a dichotomous variable, we measured the point-biserial correlation coefficient.We also detected correlations between the scores and all individual total, phospho, and cleaved proteins, using a more strict Bonferroni-adjusted P ¼ 0.01 cutoff.On top of proteins and genes directly correlated with the score, we also included their immediate neighbors in the inferred consensus coexpression networks, and any edges between them that existed in those networks.

Pathway activation profiling of the NCI-60 tumor cell lines
To understand the basal signaling network architecture of the NCI-60 panel, we conducted an initial broadscale protein pathway activation mapping analysis of 194 proteins and phosphoproteins, the largest number measured for the set to date.The full RPPA dataset can be accessed at: http://dtpsearch.ncifcrf.gov/WEBDATA_PETRICOIN_PROTEIN.zip.The results of this mapping effort are shown in Fig. 1 and revealed that clustering, composed of 3 major groups, was largely independent of the cell line origin classification (see also Supplementary Fig. S1) but was defined by the underpinning pathway activation.Other subgroups were readily apparent, composed of multiple tumor origin types and characterized by activation of several members of the EGFR superfamily as well as the downstream mTOR signaling (see also Supplementary Fig. S1).

Network module construction and analysis
On the basis of the pathway-centric nature of the signaling architecture revealed, and to more fully interrogate the relationships between phosphorylation-driven signaling at the pathway level and other "-omic" and drug sensitivity relationships, we took advantage of the fact that we measured the activation/phosphorylation of a number of key signaling proteins that spanned specific signaling pathways and developed biochemically linked signaling "modules" for systems-level analysis.We focused our "pathway module" analyses on 6 important signal transduction pathways that are central both to tumorigenic mechanisms as well as to therapeutics being evaluated in many ongoing cancer clinical trials as well as predictive marker analysis: EGFR, integrin, IGF-1R, mTOR, AKT, and apoptosis signaling (Table 1; Fig. 2 and Supplementary Fig. S2), and for which we were able to measure a large number of biochemically linked kinase substrate signaling proteins.Pathway activation scores were determined for each module such that every cell line had a continuous variable integer determinant for each pathway module.The individual proteins/phosophoproteins that were used to underpin each pathway module are shown in Table 1.In each case, we considered only the most directly involved first-order kinase-substrate relationships to minimize interactions with other signaling or pathway feedback loops.The images in Fig. 2, with the integrin pathway shown as an example, show the correlation between this ranking and the hierarchical clustering and clearly indicate which cell lines had high overall pathway activation scores and those that did not, and that these scores were driven by systemic activation of most of the biochemicallylinked individual signaling proteins.This pattern was also revealed in the other modules constructed (Supplementary Fig. S2).Interestingly, when comparing the signaling architecture derived from the pathway activation modules (Supplementary Fig. S3), a number of the lung-derived cell lines clustered together, characterized by relatively low-level  signaling activation of the analyzed phosphoproteins that comprised the modules (Table 1).

Systems level "-omic" analysis
As we defined the module-driven pathway activation states for the NCI-60 cell lines, we sought to understand all the possible relationships between our pathway module activation results and the very large and full complement of publicly available molecular data for the NCI-60 set as well as the individual RPPA-driven phosphoproteomic data we derived.In particular, the DTP serves as a vital resource for preclinical information and research materials, including web-accessible data and tools and the molecular analysis and drug sensitivity screening of the NCI-60 tumor cell line, among other information.Thus, we were able to correlate the individual RPPA phosphoprotein and protein data we generated, as well as the pathway activation scores for our 6 signal transduction networks, to DNA copy number and mutation data, methylation status, mRNA and miRNA expression, along with drug and metabolite responses.From the cell-wide protein and gene consensus coexpression networks, we selected as network nodes all of the genes and proteins that were identified as statistically correlated with the pathway activation score and linked them to the pathway score node (Fig. 3A and B and Supplementary Fig. S4).In the figures, we also included these nodes' direct neighbors in the inferred consensus networks, that is, for each node directly correlated with pathway score, we included all genes or proteins linked to that node in the inferred protein and gene coexpression networks, even if they were not significantly correlated with the pathway score.We included all edges between the selected nodes in the inferred consensus networks.For all the gene nodes in the resulting scorecentered network, we searched for genes that had significantly correlated DNA mutation, methylation, and copy number, as well as for miRNAs with correlated expression, metabolites with correlated levels, and drugs with correlated growth inhibition profiles, and included them in the network.In Fig. 3 and Supplementary Fig. S4, we used different colors to denote different types of analytes (e.g., protein, gene, metabolite, etc.), and used solid or dashed lines to indicate positive or negative correlations, respectively.For the phosphoprotein nodes, we repeated the same search for correlations using the expression of genes encoding these proteins.Also, we looked for direct correlations between phosphoprotein levels and DNA mutation, metabolite levels, and drug responses.We did not observe any RPPA endpoints that correlated negatively with pathway score, although some other types of variables (e.g., microRNA expression) with negative correlations were observed.However, the possibility of negative correlations between pathway scores and RPPA endpoints outside the pathway modules technically exists.To account for this possibility, we considered both positive and negative correlations in our analyses, and used 2-tailed statistical tests.
We also constructed a unifying joint network (Fig. 3C), attempting to link all 6 of the pathway modules together, but in this instance, we only included phosphoproteins that were significantly correlated with at least 2 different pathway scores to focus on the strongest linkages.Then, we looked for significant correlations of those phosphoproteins, or of genes encoding them, to DNA mutation, methylation, and copy number data, as well as to metabolite and drug data.For all pathway activation modules analyzed, we found that 83% (109/131) of the direct linkages to the central pathway activation score were established by individually RPPAgenerated phosphoprotein measurements (brown edges), even though we found correlations with all the other types of data.Moreover, specific statistically significant linkages were identified between RPPA-generated phosphoproteins and proteins and the other "-omic" data measured (Fig. 3 and Supplementary Fig. S4).
To assess drug sensitivity-network correlations, we considered 2 publicly available drug sensitivity databases: the A118 database and a database composed of a panel of 97 FDA-approved targeted drugs.The A118 database is composed of 118 of the most common chemotherapeutic drugs in use, and along with the 97 FDA-approved targeted drugs have been tested by DTP on the NCI-60 to evaluate their sensitivity and resistance as defined by LC 50 and GI 50 determinants.Considering all the networks together, we were able to define both positive (solid line) and negative (dash line) correlations between drug Àlog (GI 50 )s and several proteins/genes.Focusing on targeted therapies, we found significant correlations with 5 drugs specifically: imatinib, vemurafenib, sunitinib, nilotinib, and crizotinib.Interestingly, when the correlation between a drug sensitivity and a phosphoprotein was positive, the same phosphoprotein was directly and positively correlating as well with the mutation of the gene (and only with that one gene) against which the drug is specific.For example, each and every time imatinib correlated with phospho-PYK2 (or phospho-eNOS or phospho-STAT5), these endpoints correlated with the mutation of platelet-derived growth factor receptor a (PDGFRa), the known target of imatinib.This seemed to be true for every positive correlation between phosphoproteins and targeted drugs.For vemurafenib, an inhibitor of the V600E-mutated form of B-RAF, which is a component of our apoptosis, IGF-1R, mTOR, and EGFR pathway modules, we found positive correlations with phospho-p90RSK and/or phospho-PKCd (vemurafenib and phospho-PKCd correlate only in the EGFR network).These 2 phosphoproteins in turn correlated with the mutated B-RAF in the same 4 networks (10/11 NCI-60 cell lines carry the V600E B-RAF mutation; Fig. 3A and B and Supplementary Fig. S4).These results indicate that phosphoprotein levels provide a significant degree of linkage to both therapeutic sensitivity and gene mutation attributes.More extensive experimentation will be needed, using both chemical and genomic knockin/knockout, to determine the ordering and linkage of the molecular data with and between each other.

Discussion
Because the mechanism of action of nearly all oncologybased therapeutics, especially the new classes of targeted therapeutics, is based largely on the modulation of protein activity/protein expression as the principal drug target, and it is known that cancer is largely driven by disregulated and hyperactivated signaling networks (9,18), as proteomic technologies have matured, their use in these molecular characterization efforts has become increasingly important.To date, a central missing component of the NCI-60 profiling efforts has been the lack of broad-scale protein activation/phosphorylation analysis.Past integrative biology efforts that incorporated proteomic analysis of the NCI-60 set have either used a more limited number of individual proteins (N ¼ 74; ref. 10), compared with our analysis (N ¼ 135) for defined drug sensitivity analysis, or evaluated total protein levels in an attempt to link gene and protein expression levels together (and found little concordance between these datasets; ref. 6).Moreover, our own analysis of the total levels and corresponding phosphorylation levels of a number of important cytoplasmic signaling proteins such as extracellular signal-regulated kinase (ERK), AKT, and receptor proteins such as ERBB3 revealed complete lack of concordance (Supplementary Fig. S5), which indicated that direct measurements of the phosphorylation levels of a given analyte are necessary and cannot be inferred from measuring the total levels alone.Most importantly, these past efforts used a one-analyte at-a-time approach for correlation and integrated analysis, and because we now know that cancer is a pathway-oriented disease (9,18), we postulated that a more detailed molecular analysis that used a network-oriented approach would provide new insights into how different molecular compartments may link together and better characterize drug sensitivity markers.Here, we report the integration of individual protein activation/phosphorylation measurements into biochemically connected pathway modules that provide a new means to align functional protein architecture with multiple "-omic" data sets and therapeutic response correlations.
In this study, we have developed a novel systems biologybased approach to "multiomic" network analysis through the implementation of a phosphorylation-driven pathway activation score to integrate functional signaling data from RPPA analysis with all other "multiomic" data.Orientation of systems biology analysis toward pathway/networkfocused function despite individual genes, proteins, or phosphoproteins will be a critical next step in translational research.This network-based approach is useful not only as a new means to integrate genomic, metabolomic, and proteomic data together, but also to delve into the molecular basis through which a drug acts and discover new potential inhibitory mechanisms for drugs with unknown mechanisms of action.
Despite the complex relationships revealed by our analysis (Fig. 3, Supplementary Fig. S4), our approach provided an opportunity to interrogate and more fully characterize some of the specific linkages observed and provided evidence for biochemical rationale of the correlations uncovered.In several pathway activation networks (IGF-1R, EGFR, AKT, mTOR, and apoptosis) we studied, we found a consistent linkage of MST1/MST2, PAK1/PAK2, LKB1, SGK1, and ABL phosphoproteins, RUNX1T1 gene methylation status, and miRNA-211.The MST protein family is known to be involved in the Hippo pathway, a signaling cascade that controls organ size through the regulation of cell proliferation and apoptosis (19,20).As many cancers are marked by unchecked cell division, Hippo family signaling has become increasingly significant in the study of human cancers; however, how this family integrates within the context of other biochemical events still remains unclear.It has already been shown that mir-204/211 are involved in the repression of TGFbR and SNAIL signaling, and also with other genes implicated in cancer progression (21).
Another aspect of our analysis we can consider is the prevalence of miRNA correlations with gene and protein expression patterns.In particular, it was notable that many miRNAs correlated within the integrin pathway module.It is well known that miRNAs have a relevant role in several cellular mechanisms regulating the expression of genes and proteins, as well as in tumorigenesis.Within the integrin signaling network, we found that phosphorylation of cofilin has direct connections with several miRNAs reported to have roles in oncologic malignancies.For example, the ectopic expression of miR-17, -20, -93, and -106 increases proliferation, colony outgrowth, and replating capacity of myeloid progenitors and results in enhanced phospho-ERK levels (30).Meenhius and colleagues found that these miRNAs are endogenously and abundantly expressed in myeloid progenitors and downregulated in mature neutrophils and they identified sequestosome 1 (SQSTM1), a ubiquitin-binding protein and regulator of autophagy-mediated protein degradation, as a major target for these miRNAs in myeloid progenitors, showing that these miRNAs promote cell expansion, replating capacity, and signaling in hematopoietic cells by interference with SQSTM1-regulated pathways.Nishida and colleagues showed that the 2 miRNA clusters, miR-17-92a and miR-106b-25, composed of mir-17, -18a, -19a, -20a, -19b-1, -91a-1, and mir-106b, -93, -25, respectively, were upregulated in colorectal cancer stromal tissues compared with normal stroma.Gene expression profiles of the same stromal tissue samples revealed that putative targets of these miRNA clusters, such as TGFbR2, SMAD2, and BMP family genes, were significantly downregulated in cancer stromal tissue.Other downregulated putative targets were found to be involved in cytokine interaction and cellular adhesion, strengthening the evidence for involvement of these miRNAs in adhesion, including the integrin pathway (31).miR-17-92 has been shown to inhibit collagen synthesis by targeting TGFbR2, in mice (32), as well as it is known that miR-93 promotes angiogenesis and tumor growth by targeting integrinb8 (33), and together with mir-106a, seems to directly target the Cofilin2 gene (34).
The correlations we found, concerning miRNAs within both the integrin and other modules, underpin the growing relevance of these regulatory nucleic acids in governing many cellular processes.The ability to identify new miRNA targets could be enhanced by generating a systems-level analysis that could predict new, possible biologic correlations using "multiomic" datasets and bioinformatic tools.Because many investigators are excited by the potential regulatory framework of miRNA and protein expression, orienting connectivity analysis with functional protein network activation as the proximal center could provide a firm basis of such exploration.
We were also able to use our network analysis to identify correlations concerning sensitivity to specific chemotherapeutics and targeted drugs.Of particular interest was the interconnection between phosphoproteins directly connected to the central score with targeted drugs and the mutation of the gene encoding the drug target.As stated previously, we found both positive and negative correlations with 5 targeted therapeutics: imatinib, vemurafenib, sunitinib, nilotinib, and crizotinib.When these drugs had positive correlations with the gene or the phosphoprotein encoded by the gene, the same phosphoprotein was also positively correlated with the mutated gene targeted by that drug.For example, phospho-PYK2 or phospho-STAT5 correlated with both imatinib and mutated PDGFRa.Also, phospho-p90RSK correlated with vemurafenib and the mutation of B-RAF (Fig. 3A and B and Supplementary Fig. S4).Of course, in vitro experiments are needed to study the cellular responses to these drugs, and to further confirm the existence of biologic effects involving our predicted phosphoprotein-driven networks.This approach may provide a deeper understanding of how cellular biochemistry defines therapeutic response.Such "-omic" portraits could inform rational anticancer agent screenings and drive personalized therapeutic approaches.
A significant finding of the overall analysis described herein is the paucity of first-order connections to the pathway activation core with DNA genomic or RNA transcriptomic data.Most of the highly correlated analytes are either the individual phosphoproteins that comprise the activation score itself, which would be expected, or other phosphoproteins that do not underpin the score.These results indicate that gene expression and DNA mutation profiles may often not accurately predict functional protein signaling states.This has important implications in the context of personalized medicine where DNA mutation analyses are often currently used for patient stratification to drugs that target protein activity.Recent reports (35) have shown a lack of concordance of tumor mutations such as PTEN with response to inhibitors that target PI3K/mTOR signaling.Although these effects can also be underpinned by coordinate activation of compensatory pathways such as mitogen-activated protein kinase, our data show that the DNA mutation status of PTEN, while correlated with AKT activation (Supplementary Fig. S4A), has no direct correlation with mTOR pathway activation module (Supplementary Fig. S4D) in the NCI-60 cell line set.These results from our analysis are in keeping with our recent published data concerning pathway activation mapping of primary human T-cell acute lymphoblastic leukemia tumor cells, which showed no statistical linkage of PTEN DNA mutations with downstream mTOR protein pathway activation state (36), indicating that a direct measurement of signaling pathway activation is required for accurate determination of the state of the drug target.
There are certainly limitations to our current study.Because of the complexity of cell signaling and the large volume of data we integrated, we cannot simply assume that the connections we found are all clinically relevant and many likely do not occur in every patient/tumor.A critical problem for systems-level analysis of large datasets is the overfitting of the data.By using a network/signaling module-based approach, which we feel more accurately reflects the signaling architecture, we hope to minimize inaccurate conclusions because our analysis uses this as the central hub of the network and works outward.Any interconnections, however, would need to be extensively validated and verified.We also used statistical approaches that minimize overfitting, but these efforts can only dampen chances of random associations being found and not eliminate them.We could verify the correctness of these models, such as the consistent connections of the Hippo family, with in vitro chemical and genomic knockout analysis, as well as expanding our phosphoproteomic analysis to include other proteins that were not considered in the first experimental design but could be tied to these specific networks.Our approach was based on a "hub" model philosophy whereby we chose broad-scale analyses centered on interrogating signaling proteins known to be integration points within the signaling architecture, under the postulate that even with incomplete coverage, we would be measuring the aggregate signaling inputs and outputs of a larger series of proteins.By aggregating these individual protein nodes with upstream and downstream direct first-order kinase-substrate relationships, we generate more complete data about the true nature of the signaling activation state rather than just an assortment of phosphoproteins randomly distributed across the kinase space.Moreover, our analysis is based solely on data from a limited number of cell lines, which most certainly neither recapitulate the heterogeneous state of human cancer in vivo, nor are representative of all of the cancer cell line sets in the public domain.Finally, despite the broad-scale pathway activation mapping conducted, the totality of the kinome and signaling architecture was not queried and the networks chosen for exploration, although important to cancer biology and therapy, were not comprehensive to include every known pathway and drug target.Thus, we present these data as both a guide to future "-omic" analyses as well as first steps into a protein network-centered journey into future analyses rather than as a definitive dataset.
In conclusion, we used a novel, functional network-oriented approach that incorporated the most comprehensive protein pathway activation mapping analysis conducted to date on the NCI-60 cell line set to interrogate connectivity with other "-omic" data previously generated.Inclusion of the phosphoprotein data in the public domain as a growing information archive and knowledge base will provide an expanding opportunity for a systems biology view of tumorigenesis and related drug sensitivity correlations.Despite the known limitations of cell lines to recapitulate in vivo biology, cell line sets such as the NCI-60 provide a unique opportunity to investigate molecular correlations tied to causality within the defined context of the system used.Coupling extensive molecular architecture surrounding the genome, proteome, phosphoproteome, metabolome, and other molecular archives with drug sensitivity screens provide a unique "sandbox" to exploit the aggregate efforts of hundreds of scientists to more fully understand cancer biology.In the future, we hope that our pathwayoriented approach can generate new data concerning optimal therapeutic combinations, identify new predictive markers for therapeutic efficacy, and uncover new insights about how intra-and intercellular communications occur.

Figure 1 .
Figure 1.Two-way, unsupervised hierarchical clustering representing the 60 tumor cell lines (vertical axis) and the analyzed total, phospho-and cleaved proteins (horizontal axis).The clustering reveals the formation of 3 major groups (A, B, and C) that are largely independent upon the cell line origin classification but are defined by the underpinning pathway activation.Within the heatmap, red color represents higher levels of relative activity/expression; black represents intermediate levels, and green represents lower levels of relative activity/expression.The 60 tumor cell lines have been highlighted by colors indicating the tissue origin: orangekidney, bluelung, pinkbreast, redblood, greencolon, brownprostate, purpleovary, blackskin, and graycentral nervous system.

Figure 2 .
Figure 2. Identification and characterization of activated integrin pathway module by signal transduction representation (A) and unsupervised hierarchical clustering (B).For the pathway representation, the inhibitory phosphorylations considered in the analysis are shown in red and the activating phosphorylations in green.For the unsupervised hierarchical clustering, the complete panel of NCI-60 is shown (vertical axis).Specific endpoint relative intensity values have been used to create the heatmap.After overall score calculation, we highlighted the cell lines with the top 10 pathway scores (red), and the cell lines with the lowest 10 pathway activation scores (green).

Figure 3 .
Figure 3. Systems networks of EGFR (A) and integrin (B) pathway activation, together with a unified pathway activation network (C).Node color: Violet, node representing the pathway score; Blue, phosphoproteins (linked to pathway score or to other proteins/genes at the protein level); Yellow, genes (linked to pathway score or to other proteins/genes at the mRNA level); Orange, miRNA; Dark green, drug; Light green, metabolite; Gray, other.Node shape: Diamond, pathway score node, or phosphoprotein that is used in calculating the pathway score; Circle, other nodes.Edge color and label: Brown, relationship inferred on the basis of phosphoprotein level (either with level other phosphoprotein or with pathway score); Gray, relationship inferred on the basis of gene mRNA expression (either with mRNA of other gene or with pathway score); Dark green, phosphoprotein level or gene expression (mRNA) is significantly correlated with drug response measured as Àlog (GI50); Light green, phosphoprotein level or gene expression (mRNA) is significantly correlated with metabolite concentration; Red, phosphoprotein level or gene expression (mRNA) is significantly correlated with mutation of other gene.Arrow points from mutation gene to the mRNA gene; Pink, gene expression (mRNA) is significantly correlated with methylation of other gene.Arrow points from methylation gene to the mRNA gene; Dark blue, gene expression (mRNA) is significantly correlated with copy number of other gene.Arrow points from copy number gene to the mRNA gene; Orange, gene expression (mRNA) is significantly correlated with expression of microRNA.Edge line style: Solid, positive correlation; Dashed, negative correlation.

Figure 4 .
Figure 4. Hippo-based model.Arrows point out the direction of signaling: in orange, the YAP signaling effects on p73 driven by c-Abl, in blue the YAP signaling effects on the transcription factor RUNX1 driven by c-Src.In pink, the involvement of mir-211 in stressactivated protein kinase (SAPK)/cjun-NH2-kinase (JNK) and SMAD signaling, as well as in epithelialmesenchymal transition (EMT) is shown.Signaling pathway model made with PathVisio v2.0 (37).