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Pathway-Centric Integrative Analysis Identifies RRM2 as a Prognostic Marker in Breast Cancer Associated with Poor Survival and Tamoxifen Resistance123

Breast cancer (BCa) molecular subtypes include luminal A, luminal B, normal-like, HER-2–enriched, and basal-like tumors, among which luminal B and basal-like cancers are highly aggressive. Biochemical pathways associated with patient survival or treatment response in these more aggressive subtypes are not well understood. With the limited availability of pathologically verified clinical specimens, cell line models are routinely used for pathway-centric studies. We measured the metabolome of luminal and basal-like BCa cell lines using mass spectrometry, linked metabolites to biochemical pathways using Gene Set Analysis, and developed a novel rank-based method to select pathways on the basis of their enrichment in patient-derived omics data sets and prognostic relevance. Key mediators of the pathway were then characterized for their role in disease progression. Pyrimidine metabolism was altered in luminal versus basal BCa, whereas the combined expression of its associated genes or expression of one key gene, ribonucleotide reductase subunit M2 (RRM2) alone, associated significantly with decreased survival across all BCa subtypes, as well as in luminal patients resistant to tamoxifen. Increased RRM2 expression in tamoxifen-resistant patients was verified using tissue microarrays, whereas the metabolic products of RRM2 were higher in tamoxifen-resistant cells and in xenograft tumors. Both genetic and pharmacological inhibition of this key enzyme in tamoxifen-resistant cells significantly decreased proliferation, reduced expression of cell cycle genes, and sensitized the cells to tamoxifen treatment. Our study suggests for evaluating RRM2-associated metabolites as noninvasive markers for tamoxifen resistance and its pharmacological inhibition as a novel approach to overcome tamoxifen resistance in BCa.


Introduction
In the United States, breast cancer (BCa) is the most common cancer diagnosed in women and the second highest cause of cancer-related deaths among them [1]. Once diagnosed, many important characteristics of BCa are used to determine optimal treatment and prognosis. These characteristics include tumor size, estrogen and progesterone receptor status (ER and PR), HER-2/neu status, histologic subtype, nuclear grade, lymph node status, and margin status [2,3], all of which provide limited insight into the molecular pathways driving disease progression. Breast tumors are clinically stratified into subgroups on the basis of ER and HER-2 expression and the so-called triple-negative tumors (TN: ER, PR, and HER-2 negative) for which currently there is no targeted therapy. Hence, TN subtype tumors are often treated using conventional chemotherapeutics [4,5].
To obtain a better understanding of the pathways associated with estrogen-induced molecular alterations, numerous studies have examined gene and protein expression profiles using high-throughput omics-based technologies [6][7][8][9][10][11][12][13][14][15][16][17][18]. However, the application of metabolomics to define pathways associated with BCa has been limited. Unlike the genome and the proteome, the metabolome defines the actual physiological state of the tumor, is computationally tractable, less complex (than the other -omics), and more importantly, reveals potential metabolites that can be measured in noninvasive body fluids in a clinical context. Some researchers have used mass spectrometry to examine the metabolome associated with BCa [19][20][21] as well as to determine altered metabolites and biochemical pathways associated with the various subtypes of tumors [22][23][24][25][26][27][28].
In the current study, using a robust mass spectrometry platform [29][30][31][32], we measured metabolic alterations in luminal and basal BCa cell lines [33] and ranked pathways using a Gene Set Analysis (GSA)-based enrichment approach [34]. The enriched pathways were then selected on the basis of their relevance in patient-derived luminal and basal-like BCa tissues, by examining pre-existing gene and metabolic expression data sets. Following this, the selected pathways were further stratified on the basis of their association with survival of patients with BCa using publicly available gene expression data sets containing information on patient outcome. A novel rank-based method was developed that took into account the degree of enrichment of the pathways in each of the molecular data sets, as well as its prognostic potential, to generate a cumulative rank score. This was then finally used to stratify the pathways for subsequent downstream validation studies. This systematic stepwise selection enabled us to identify pyrimidine metabolism as a key biochemical pathway associated with aggressive BCa in general and with tamoxifen resistance in patients with luminal BCa. Importantly, using in vitro and in vivo BCa models, the translational and clinical relevance of pyrimidine metabolism and the gene associated with one of its key enzymes, ribonucleotide reductase subunit M2 (RRM2) was established.
For studies to characterize the role of RRM2 in tamoxifen resistance, MCF-7 parental without treatment (MCF-7 L, parental) or either treated with tamoxifen for 48 hours [when cells are still sensitive, TAM sensitive (TAM-S)] or for long term until cells became resistant and resumed growth [TAM resistant, TAM-R)] as described earlier by Morrison et al. [35,36] were used. The parental Neoplasia Vol. 16, No. 5, 2014 Integration of Omics Data in Breast Cancer Putluri et al.
cells were grown in RPMI medium as described above. Tamoxifentreated cells (TAM-S or TAM-R) were grown in phenol-red-free RPMI medium (Media Tech, Manassas, VA) containing charcoal stripped Fetal Bovine Serum, with 10 uM 4-hydroxytamoxifen (Sigma-Aldrich, St Louis, MO). In addition, xenografts generated using MCF parental cells (MCF-7 L) that were either untreated (termed parental) or treated with tamoxifen for~2 weeks (short-term treatment and TAM-S) or~3 months (long-term treatment and TAM-R), as described by Massarweh et al. were used.

Mass Spectrometry
Unbiased mass spectrometry-based methods were described earlier [30]. Sample preparation for mass spectrometry-based examination of metabolome is described in Supplementary Methods. The mass spectrometry portion of the unbiased profiling platform is based on a 1200 SL Rapid resolution LC and a 6520 Quadrupole Time of Flight mass spectrometer (Agilent Technologies, Santa Clara, CA). For unbiased profiling studies, real-time mass correction during mass spectrometry was achieved by infusion of a standard mixture of reference ions using an independent 1200 SL Rapid resolution LC isocratic pump equipped with 100:1 splitter to output a flow rate of 5 ml/min. The samples were independently examined in both positive and negative ionization modes using a dual electrospray ionization source. Detailed description of chromatographic methods used for separation of metabolites is given in Supplementary Methods.
The mass spectrometry portion of the targeted profiling platform was based on a 1200 SL Rapid resolution LC and a 6430 triple Quadrupole mass spectrometer (Agilent Technologies). The multiple reaction monitoring (MRM)-based measurement of levels of 76 metabolites was done using four different methods, which used either a reverse-phase or normal aqueous-phase chromatographic separation, before mass spectrometry. Details of the methods, associated chromatographic conditions, metabolites measured, and their corresponding MRM transitions are given in Supplementary Table 2

Metabolomic Library, METLIN
METLIN (Agilent Technologies) was used to search the unbiased mass spectral data. METLIN was created using approximately 1800 commercially available compounds whose retention time was defined using the Reverse Phase (RP) chromatographic method described above.

Metabolomics Data Analysis
All the downstream processing and data analyses were performed using R statistical programming software (R Foundation for Statistical Computing, Vienna, Austria) [37]. After removing the compounds with more than 40% of missing values, the missing values in the remaining metabolites in the BCa cell lines were imputed using the K-Nearest Neighbour (KNN) algorithm ("imputation" package [38], K = 5). Following the preprocessing, of 673 compounds, the BCa cell line has 76 were unique metabolites, whereas our recently published BCa tissue metabolome data set [39] had a total of 219 compounds of which 168 metabolites were named. Imputed data were median centered and Inter Quartile Range (IQR) scaled following log 2 transformation. Two-sided t tests were performed to identify differential metabolites by comparing luminal and basal subtypes coupled with False discovery rate (FDR) adjustment (adjusted P values b .2) using the Benjamini Hochberg (BH) method [40] along with estimated fold change using the Differential Expression via Distance Summary (DEDS) package [41].

Analysis of Microarray Gene Expression Data
In this study, we used this gene expression data set (termed National Cancer Institute data set, GSE37751) for 46 matched BCa tissues derived from (Affymetrix, Santa Clara, CA, USA) GeneChip Human Gene 1.0 ST arrays, followed by Robust Mult-chip Average (RMA) normalization [39,42] found in the R bioconductor [43] package and differential expressions analysis using FDRadjusted (Benjamini Hochberg) two-sided t tests (limma (linear models for microarray data) package). Heat maps with average linkage based hierarchical clustering of z-score transformed differentially expressed compounds and genes, were generated using the gplots [44] package.

Identification of Key Pathways Based on Enrichment Scores Describing Differential Expression and a Prognostic Value in BCa Subtypes
Selection of key pathways was accomplished using a two-step process that considered both the relative enrichment scores on the basis of a modified enrichment analysis, GSA (proposed by Tibshirani et al. {(http://www-stat.stanford.edu/~tibs/GSA/), [45,46]} and their prognostic potential on both the set of metabolites and their Kyoto Encyclopedia of Genes and Genomes (http://www.genome.jp/kegg/) (KEGG)-derived associated gene sets across multiple publically available BCa gene expression data sets (refer to Supplementary Table  3 for the list of metabolites and their associated genes and Supplementary Table 4 for the description of the data sets).
In the first step, the selection of top significant (enrichment P value) pathways included a group of 11 pathways that were altered between basal and luminal subtypes. Next, the gene sets associated with these 11 enriched pathways were obtained from KEGG to derive their pathwayspecific score (averaged gene expression) and examined for their prognostic value in the publically available gene expression data sets (refer to Supplementary Table 4 for the list of data sets used) [47,48]. This was done by examining the association between pathway scores and years of metastasis-free patient survival using a Cox proportional hazards model. For selected pathway-associated gene sets or individual genes, correlation with metastasis-free survival was visualized using Kaplan-Meier (KM) plots. As a comparative reference for the prognostic analysis, samples in the Kessler compendium and van de Vijver data sets were also stratified using (GEO data set GEICAM9906) PAM50 and other known prognostic marker panels [49].
Once the enrichment scores for both expression levels and prognostic values were obtained for each pathway, they were combined to generate a combined rank (CR) score, details of which are described in Supplementary Methods.

Analysis of RRM2 Expression by Immunohistochemistry on Tissue Microarray of Patients with BCa
Tissue microarray construction. RRM2 protein expression was measured using a previously developed tissue microarray (TMA) (Pathology Resource Network, Roswell Park, NY) containing 192 biopsy specimens (each having three replicates). Tissues from patients who had surgeries performed between 1995 and 2008 at Roswell Park Cancer Institute (Buffalo, NY) were included in the TMA. Specimens for controls within the TMA consisted of multiple cores of normal tissue from 10 different organs including heart, colon, kidney, adrenal, ovary, myometrium, brain, thyroid, lung, and prostate, thereby representing more than 20% of all the cores in a TMA. Appropriate Institutional Review Board approval consistent with federal, state, and local requirements was obtained for this project, and clinical and outcome data were deidentified.
Of the 192 patients whose tissues were arrayed on the TMA, 185 were ER positives, 5 were ER negatives, and the remaining 2 were not defined for their subtype. Further among the 192 tissues, 132 were PR positive, 58 were PR negative, and the remaining two patients were not defined for their PR status. Patients were administered with tamoxifen (156), anastrozole (21), or letrozole (2) or not treated (5). The treatment was given either in an adjuvant systemic setting (165) or neoadjuvant systemic setting (16). Two patients received systemic treatment for metastasis, one received for local recurrence, four did not receive any systemic treatment, one patient refused treatment, and three patients were not documented for the treatment modality. In addition, a subset of patients were also subjected to chemotherapy with the following distribution: Importantly, in this study, the inclusion criteria for evaluating RRM2 expression in the TMA were that the patient should have ER + tumors, be treated with tamoxifen in an adjuvant setting and without chemotherapy. Using these criteria a total of 45 patients were selected, of which recurrence was seen in 19 patients whereas the rest of the 26 patients were followed for recurrence-free survival for a median time of 8 years. Furthermore, among these 45 patients, 25 patients were reported to have died by the end of 8 years of median follow-up, of which 13 died of the disease, 6 died due to other complications, and 6 died of unknown causes.

Immunohistochemistry
Paraffin sections were cut at 4 μm, placed on charged slides, and dried at 60°C for 1 hour. Slides were cooled to room temperature, deparaffinized in three changes of xylene, and rehydrated using graded alcohols. For antigen retrieval, slides were heated in the steamer for either 40 or 60 minutes in citrate buffer (pH 6.0) (Biocare Medical, (Concord, CA) No. CB910), followed by a 20-minute cooldown. Endogenous peroxidase was quenched with aqueous 3% H 2 O 2 for 10 minutes and washed with PBS/T (Tween-20). Slides were loaded on a Dako (X0909, Dako, CA) autostainer, serum-free protein block (Dako No. X0909) was applied for 5 minutes and blown off, and the RRM2 antibody (SC-81850, Santa Cruz Biotechnology (Santa Cruz (Dallas, TX)) was applied at 1:500 dilution for 1 hour. PBS/T was used to wash slides between each reagent application. Dako Mouse Envision (K4007) was applied for 30 minutes, followed by DAB (Dako No. K3468) for 10 minutes. Finally, the slides were removed from the autostainer, counterstained with hematoxylin, dehydrated, cleared, and coverslipped. Isotype-specific nonimmune IgG was used as a control to examine the specificity of RRM2 staining.

Aperio Slide Scanning and Image Analysis
TMA slides were digitally scanned using Aperio ScanScope (Aperio Technologies, Inc, Vista, CA) with × 20 bright-field microscopy.
These images were then accessible using Spectrum (Aperio Technologies, Inc), a web-based digital pathology information management system. Slide images were automatically associated to a digital slide created in the Digital Slide table in Spectrum. Once slides were scanned, Aperio ImageScope version 11.2.0.780 (Aperio Technologies, Inc) was used to view images for analysis. Slide image data fields were populated and images were examined for quality and were amended as necessary. Care was taken to avoid including areas of carcinoma in situ and regions with staining artifacts. When possible, representative areas of tumor were selected for analysis with a minimum target of 30 tumor cells per TMA core.
A Cytoplasm Algorithm (Aperio, Leica Microsystems, IL) that was calibrated to analyze DAB staining intensity and calculate the percentage of cells containing the stain within their cytoplasmic compartment was used. Staining thresholds were set for calling out positive stains, and the scores for average cytoplasm intensity for the selected regions were calculated on the basis of these thresholds. The staining intensity was stratified into the following four score values: 0, none; 1 +, weak; 2 +, moderate; and 3 +, strong. Concomitantly, for each of these staining scores, the percentage of cells that stained was also calculated. The staining intensity and the percentage of cells in each of the staining intensity bins were together used to calculate the H score that reflects the proportion of cells in the cytoplasm of the tumor that express RRM2 at the various staining intensity thresholds described above. In other words, the H score was calculated as follows: 1*(%1 +) + 2*(%2 +) + 3*(%3 +), and ranged from 0 to 300 with a score of 300 reflecting 100% of the cells having a staining intensity of 3 +. All the slides were counterstained with hematoxylin to reveal the morphologic detail of the surrounding tissue and to help identify nuclear and cytoplasmic compartments of the cells for analysis.

TMA Data Analysis
In this study, we selected tissues from the TMA for RRM2 analysis that met the following inclusion criteria: ER + treated with tamoxifen in the adjuvant setting and no chemotherapy. A total of 45 tissues passed these criteria among which 19 recurred and the rest did not show recurrence for a median follow-up time of 8 years. Furthermore, among the 45 patients, 25 were reported to be dead by the end of the follow-up period either due to cancer (13) or other causes (12).
For the analysis, the median H score value for each of the samples was calculated using data from replicate cores. The median H score was then compared using nonparametric rank sum test for RRM2 expression among patients who were reported to be dead versus those reported to be alive for the 8-year median follow-up time period. Furthermore, KM plots using H score were used to examine the association of RRM2 with recurrence-free survival.

Genetic and Pharmacological Inhibition of RRM2 in TAM-R Cells and Associated Characterization
TAM-R cells were transfected using either negative control siRNA (ctrl-siRNA) small interfering Ribo Nucleic Acid (siRNA) or Smart pools of siRNA against RRM2 (RRM2-siRNA, Thermo Scientific/ Dharmacon RNAi Technologies, Chicago, IL) using Lipofectamine 2000 reagent as described earlier [50]. At 48 hours of posttransfection, whole-cell extracts were made, and knockdown of RRM2 was verified using both Quantitative Polymerase Chain Reaction (QPCR) and immunoblot analysis. Bromodeoxyuridine (BrdU) labeling assay (Calbiochem/Millipore, Billerica, MA, USA) was used to assess the proliferation rate per manufacturer's instructions. For cell-cycle gene Neoplasia Vol. 16, No. 5, 2014 Integration of Omics Data in Breast Cancer Putluri et al.
analysis studies, TAM-R cells were transfected with ctrl-siRNA or RRM2-siRNA as described above. After 48 hours, total RNA was isolated from TAM-R cells, and cDNA was prepared from total RNA using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA). Transcript levels of cyclin genes were measured by performing qPCR using SsoAdvanced universal qPCR supermix and gene specific primers as per manufacturer's instructions (Bio-Rad Laboratories). For 5-azacytidine (aza)-based studies, TAM-R cells were plated onto 96well and six-well plates for assessing cell survival and RRM2 protein levels, respectively. The next day, cells were treated with control vehicle or aza at 25, 50, and 100 nM concentrations. Following this, after 72 hours, cells were subjected to MTT (Non-Radioactive Cell Proliferation Assay (Promega Corporation, Madison, WI)) assay and immunoblot analysis.
Results Figure 1A shows the overall approach used in this study. Mass spectrometry was used to measure the relative levels of 673 compounds (includes 60 metabolites measured using targeted assays) across 10 BCa cell lines (luminal n = 4 and basal n = 6; see Supplementary Table 1 for description of cell lines), each analyzed as biologic triplicates. Metabolites from predefined liver pools (n = 26) were extracted in parallel and examined as process controls. Internal standards were spiked in during the extraction B A Figure 1. Metabolomic profiling of BCa cell lines. (A) Overview of metabolomic profiling and the integrative methodology. Mass spectrometry-based measurement of the cell line metabolome was followed by GSA-based identification of enriched biochemical pathways. These were compared with GSA-enriched metabolic pathways derived from an independent set that consisted of matched metabolome and transcriptome data derived from~50 BCa tissues (46 specimens for transcriptome data). Pathway enrichment results from each of the three data sets were integrated using a novel rank-based method to nominate a set of 11 commonly enriched pathways. These were further curated using a similar rank-based approach for their clinical significance, i.e., association with time to metastasis-free survival. Overall, results of this stepwise enrichment were used to nominate pathways on the basis of both their biologic and clinical relevance. (B) Heat map overview of levels of named metabolites across luminal and basal/mesenchymal BCa cell lines. Shades of maize and blue represent elevated and reduced levels of metabolites, respectively (see color scale). process to assess process variation. Mass spectral data were used to calculate differential metabolites that distinguish basal and luminal BCa cell lines. To obtain a pathway perspective, the metabolite data were enriched using GSA [34]. In parallel, the same strategy was used on matched pre-existing BCa tissuederived gene expression and metabolic data to determine clinically relevant biochemical pathways. Subsequently, commonly enriched pathways in both the cell line and patient data sets were determined and evaluated for their association with BCa-specific survival. A novel rank-based scoring method incorporating information on both the biologic (enrichment in clinical tissue data sets) and clinical (prognostic) significance of the pathways was developed and employed to select key biochemical pathways for downstream validation studies. This method allows us to define key biochemical pathways associated with cancer progression, by integrating data from diverse omics data sets containing well-annotated clinical data.

Metabolic Profiles Associated with BCa Cell Lines
Before the analysis of the cell line data, the process controls, i.e., matrix-free internal standards and liver pools were evaluated for their variability. The range of coefficient of variation (% CV) for the eight internal standards in liver pool was 0.09% to 6%. A total of 76 named metabolites were measured across the luminal and basal BCa cell lines (Supplementary Figure 1 and Supplementary  Table 1). Of these, 42 metabolites (FDR-adjusted P value b .2) were differentially altered between the luminal and basal subtypes ( Figure 1B). Luminal BCa cell lines showed elevated levels of amino acids, like phenylalanine, tryptophan, and tyrosine, and branched chain amino acids like leucine, lysine, and valine, as well as higher levels of lauric acid and oleic acid. In contrast, levels of nucleotides like guanine, adenine, thymine, uracil, xanthine, and guanosine were elevated in basal BCa cells compared to the luminal counterparts.

Enrichment Analysis to Define Pathways Associated with Cell Line Metabolomics Data
To obtain better insight into the subtype-specific biochemical pathways distinguishing luminal from basal BCa cell lines, we applied GSA on the cell line-derived metabolomics data as described under the Methods section. As a first step in this process, all the named metabolites identified in cell lines (n = 53) were mapped to 200 biochemical pathways using the KEGG database (Supplementary Table 2) [51]. These pathways were then used for GSA-based enrichment analysis. Following this, we ranked the pathways on the basis of their FDR-corrected enrichment P value and selected 30 pathways (P value b .15; Supplementary Table 5) for subsequent integrative analysis with pathways obtained from tissue data sets. The enriched pathways in the cell line data include those that describe metabolism of purine, pyrimidine, glutamate, alanine-aspartate, glycine-serine-threonine, butanoate, β-alanine, taurine-hypotaurine, valine-leucine-isoleucine biosynthesis, pantothenate-CoA (Coenzyme A) biosynthesis and lysine degradation.

Enrichment of Biochemical Pathways in Luminal Basal-Like BCa Tissues
We used matched transcriptomic and metabolomic data sets for human breast tumors that were generated earlier by our group [39]. Metabolomics data were available for a total of 50 matched benign-tumor pairs that included 33 luminal and 17 basal-like/TN tumors. Here, a total of 219 compounds were measured, of which 98 metabolites were mapped to~200 biochemical pathways in KEGG. Supplementary Figure 3A shows the overall heat map for the metabolites in luminal and basal-like BCa tissues. A total of 44 pathways were enriched (P value b .15) using GSA on the tissue metabolic data set that were subsequently ranked on the basis of their P value and used for integrative analysis with cell linederived biochemical pathways (Supplementary Table 5 lists the enriched pathways).
The transcriptomic data, published by our group earlier [39], contained measurements for~20,000 genes that were measured using Affymetrix ST arrays across 46 BCa tissues (ER + n = 31 and basal like/TN, n = 15; see Supplementary Figure 3B, GEO Accession No. GSE39004/GSE37751). Among these,~2100 genes were mapped to the same 200 KEGG biochemical pathways described above and used for GSA-based enrichment analysis. Supplementary Table 5 shows the list of the 69 pathways enriched using the gene expression data (P value b .15) that distinguish luminal versus basal BCa. These pathways were also ranked on the basis of their FDR-corrected enrichment P value and used for subsequent integrative analysis.

Selection of Robust Omics-Derived Pathways
Having determined the enriched biochemical pathways and their respective ranks in the metabolome (cell line and tissue) and transcriptome (tissue) data sets, we next asked which of these metabolic processes were consistently enriched across all the three data sets. A total of 11 pathways distinguishing luminal versus basal BCa were uniformly enriched at a P value threshold b .15 in each of the three data sets (Figure 2, A-C). These 11 pathways included those that describe metabolism of purine, pyrimidine, glutamate, alanine-aspartate, glycine-serine-threonine, butanoate, β-alanine, taurine-hypotaurine, valine-leucine-isoleucine biosynthesis, pantothenate-CoA biosynthesis, and lysine degradation (Supplementary Figure 2). Supplementary Figure 3 shows the heat maps for the expression of the genes associated with the 11 biochemical pathways in luminal and basal breast tumors from our BCa tissue-derived transcriptomic data set [39].

Integration of Omics-Based Enrichment and Prognostic Ranks to Select Clinically Relevant Biochemical Pathways
The biochemical pathways that were consistently enriched across the different omics data sets were further filtered for their clinical relevance by examining their prognostic potential in publicly available BCa gene expression data sets having at least 10 years of clinical follow-up information. An in silico analysis was performed to investigate the association of each of the 11 metabolic pathways with patient survival using 10 independent gene expression data sets (refer to Supplementary Table 4). Among these, eight data sets (all on Affymetrix platform) are included in the Kessler compendium [48]. The additional two data sets include the van de Vivjer data set (containing gene expression data on 295 patients with BCa [47] and the NCI transcriptome data set (GEO data set GSE39004) (used for integrative analysis above [39]. For the analysis, both the expression value and directionality of expression for each gene associated with the 11 commonly enriched pathways (described in Figure 2, A-C) were obtained from each of the above gene expression data sets. This information was then used to generate an average gene expression score for each pathway, which was then examined for association with years of metastasis-free patient survival using a Cox proportional Neoplasia Vol. 16, No. 5, 2014 Integration of Omics Data in Breast Cancer Putluri et al.
hazards model. The prognostic value of the top ranked pathway was visualized using KM plots across all samples in the Kessler compendium. Figure 2D shows the individual as well as integrated P values for each pathway obtained across all BCa tissues using all the 10 data sets described above. Notably, only pyrimidine metabolism had a P value b .1 for the CR Score, indicative of its biologic and clinical relevance in BCa (Table 1). In addition, pyrimidine metabolism was also significantly enriched in a subset of BCas with documented ER status (Supplementary Table 6). Consistent with all of this, KM plots also showed higher expression of pyrimidine metabolism pathway genes as being associated with shorter metastasis-free survival, across all BCa ( Figure 2E, N = 1340, log-rank P value = .0004) as well as within the subset of ER + tumors ( Figure 2F, N = 686, log-rank P value = .003). In addition, higher expression of pyrimidine metabolism genes, correlated well with PAM50 [52], defined basal-like, Her-2-enriched, and luminal B tumor subtypes whereas being lower in luminal A tumors ( Figure 2G). Higher cumulative expression of pyrimidine Integration of Omics Data in Breast Cancer Putluri et al. Neoplasia Vol. 16, No. 5, 2014 metabolism genes also overlapped with expression patterns of MKi67 [53], Oncotype Dx [54], Mammaprint [55], and genome grade [56] ( Figure 2G). In contrast to the above, genes belonging to the methionine and glutathione pathways showed opposing expression patterns in basal-like and Her-2-enriched tumors ( Figure 2G).

Higher Expression of RRM2 is Associated with Tamoxifen Resistance in BCa
We examined the genes within the pyrimidine metabolic pathway that were most highly altered in basal versus luminal BCa using our tissuederived transcriptome data [39] Both uridine pyrophosphorylase 1 (UPP1, Supplementary Figure 4) and RRM2 ( Figure 3A) were significantly (P = .0004) elevated in basal-like BCa compared to luminal tumors. Ribonucleotide reductase has three isoforms, M1 (RRM1), M2 (RRM2), and M2b (RRM2b), all of which are key enzymes in the process of DNA replication and catalyzes the conversion of UDP and cytidine diphosphate (CDP) to their respective deoxygenated versions (dUDP/dCDP), which are then used for DNA synthesis. Furthermore, higher levels of RRM2 but not RRM1 (refer to Supplementary Table 7) were associated with poor survival in the Kessler compendium (log-rank P = 3.6E-09; Figure 3B) as well as the Wong, Loi, and van de Vivjer data sets, each of which contains a significant number of luminal A patients ( Figure 3C) [47,57] who are conventionally treated with endocrine or hormone therapy. In light of the above finding, we asked whether RRM2 could distinguish luminal patients on the basis of their response to tamoxifen (TAM), a widely used endocrine agent for ER-positive BCa.
Our analysis using the Loi data set (that contains 149 ER + node-negative patients treated with TAM), showed that RRM2 by itself was able to distinguish patients who exhibit intrinsic resistance to TAM treatment and hence relapse within 2 to 5 years after starting the therapy (log-rank P value = .002; Figure 3D).
Consistent with this, a KM plot ( Figure 3D) showed that about 70% of the patients who had low expression of RRM2 (bottom third) in this data set showed significantly longer metastasis-free survival, compared to about 50% who expressed higher levels of this pyrimidine metabolic gene (top third) and displayed worse outcome. Furthermore, RRM1 was not altered between tamoxifen responders and nonresponders in this data set, whereas RRM2b was not measured. Consistent with all of the above, in the Loi data set, pathway-centric enrichment of gene expression data stratifying patients into TAM responders versus nonresponders also significantly enriched pyrimidine metabolism (Supplementary Table 8).
We next verified the association between RRM2 overexpression and TAM resistance using TMA. Here, we restricted our analysis to ER+ BCa tissues from patients treated with only TAM in the adjuvant setting and followed up for tumor recurrence and survival for a median time period of 8 years. To begin with, the specificity of RRM2 antibody for the protein was established using an isotype nonimmune IgG control (Supplementary Figure 5). Following this, TMA staining using RRM2 antibody revealed significantly higher expression of the protein in patients who did not respond to TAM ( Figure 3E). Consistent with this, patients who were reported to have died by the end of the follow-up  Neoplasia Vol. 16, No. 5, 2014 Integration of Omics Data in Breast Cancer Putluri et al.
period expressed significantly higher RRM2 (Wilcoxon rank sum P = .04) compared to those who reported to be alive ( Figure 3F). In addition, higher RRM2 expression associated significantly with earlier tumor recurrence (log-rank test, P = .04; Figure 3G), confirming the prognostic value of RRM2 in patients with luminal BCa treated with TAM. Next, to determine the role of RRM2 in TAM resistance, RRM2-associated metabolites were measured in MCF-7 L cells that were treated with TAM either for a short-term (TAM-S) or longer time period (TAM-R) [58,59], as well as xenograft tumors [58] generated using MCF-7 L cells that were treated in vivo with TAM for 2 weeks (TAM-S) or 3 months (TAM-R). As expected from our earlier findings, in both cell lines and xenograft tumors, the ratio of the metabolite product:metabolite substrate for RRM2 (dUDP/UDP and dCDP/ CDP) were significantly higher in TAM-R cells (P b .01; Figure 4, A and B, TAM-Cell lines and Figure 4, C and D, TAM-Xenograft),compared to TAM-S controls. A similar profile was also obtained for TAM-R cells when compared with parental untreated controls (MCF-7 L, Figure 4, A and B). Corroborating these findings, both protein ( Figure 4E; refer to Supplementary Figure 6 C and D, C and D, for data using untreated parental cells) and mRNA levels ( Figure 4F) of RRM2 were higher in TAM-R cells compared to TAM-S controls. Furthermore, transcript levels of RRM2 were also elevated in TAM-R xenograft tumors compared to TAM-S counterparts (Supplementary Figure 7). To further substantiate the association of RRM2 with TAM resistance, siRNA-based knockdown of the gene was carried out (immunoblot for Knock Down (KD) in Figure 4G) in TAM-R cells. As expected, siRNA-mediated KD of RRM2 expression in the resistant cells resulted in a significant decrease in cell proliferation compared to nontarget siRNA controls ( Figure 4H). Consistent with this, the transcript levels for cyclins regulating G 1 -S transition Cyclin D2 (CCND2) and Cyclin E1 (CCNE1) were significantly (P b .05) reduced in the KD cells with no change in the mRNA and levels of Sphase-or G2-phase-associated cyclins (CCNA1 and CCNB1) ( Figure 4I).
Having garnered substantial evidence on the association of RRM2 with TAM resistance, we next evaluated the ability of the DNA methyltransferase inhibitor aza to sensitize TAM-R cells to TAM treatment. This was motivated by a recent report that showed downregulation of RRM2 transcript and protein levels by aza in leukemic cell lines [60]. Consistent with the reported findings, treatment with 25 to 100 uM aza completely abolished RRM2 protein expression in TAM-R cells ( Figure 4J). Furthermore, aza-treated TAM-R cells showed a significant reduction in the cell proliferation as assessed using BrdU assay ( Figure 4K).

Discussion
To delineate the biochemical processes altered in BCa, we adopted a strategy wherein we started by defining the metabolic alterations in luminal and basal-like BCa cells that have been well characterized for their subtype gene expression and routinely used in laboratory studies [33]. The metabolic profiles were analyzed to generate pathways that were then examined for their relevance using clinical specimens. The caveats that argue against using cell line models for profiling studies include the alteration in their molecular profiles caused by culture conditions [61] and the lack of the intratumoral heterogeneity in cell lines [62]. In contrast, the challenge of using clinical specimens for translational research lies in interpatient variability as well as the heterogeneous nature of tumor-associated cell types. In spite of these potential confounders, it is encouraging to find a subset of metabolic pathways that were consistent between cell lines and patient tumors and distinguish luminal from basal-like BCa. The novelty of our integrative approach lies in identifying these commonly altered biochemical pathways using a pathway-centric rank-based method that takes into account both the degree of enrichment (which is a reflection of their biologic importance) of the pathway in patient-derived omics data and its association with patient prognosis (reflection of their clinical relevance). In this regard, our methodology is distinct from the ones that rely on either a single data type (for example, gene expression, [63]) or combined data sets using concordance-based integrative methods [64]. However, our approach is comparable to the metabolome-centric method described by Imielinski et al. [65], with refinements that incorporate a strong patient-centric connotation to calculate the CR Score. This is exemplified in our results, wherein pyrimidine was ranked as the top pathway among the 11 possible contenders on the basis of both its quantity in patient tumors and its association with patient's clinical outcome. Pyrimidine metabolism signifying the proliferative ability of tumor cells was also reflected by its elevated presence in PAM50-defined basal, Her-2-enriched and luminal B tumors. RRM2 is a key gene in pyrimidine metabolism and has been earlier shown to be elevated in aggressive BCa [66]. In combination with other proliferative markers, RRM2 has also been found to have prognostic relevance in BCa [67]. Consistent with this, in our study, RRM2 expression by itself was able to distinguish good versus poor survivors within the entire group of patients with BCa that included a significant proportion of luminal A subtype. Importantly, from the clinical standpoint, luminal A patients are typically considered to have a better survival outcome. The clinical value of molecular predictors like PAM50 [68], Oncotype Dx [54], Mammaprint [47], and other prognostics stems from their ability to distinguish a subset of aggressive tumors within this so-called clinically indolent patient population. In light of this, it is remarkable to note that the prognostic value of RRM2 alone was comparable to all the above panel of markers, setting the stage for future prospective validation of this gene in independent patient specimens. Furthermore, in the setting of tamoxifen treatment that is routinely administered to ER + patients, RRM2 was able to distinguish patients who were resistant to treatment from those who responded to the therapy. This is verified by higher expression and activity of RRM2 in TAM-R cell lines and xenograft models [58,69]. Furthermore, a significantly larger proportion of TAM-treated patients who died of BCa showed higher RRM2 protein expression, a novel finding that was further validated using TMA. Importantly, this finding does not imply a causal role for RRM2 in the onset of TAM resistance. Furthermore, the findings on higher RRM2 expression both in tumors with poor prognosis irrespective of the subtype and in TAM-R ER + tumors warrant additional studies to establish the role of the protein in these patient populations.
RRM2 belongs to the family of ribonucleotide reductase that has two other isoforms, RRM1 and RRM2b. The three together catalyze the conversion of uridine/cytidine containing nucleotide triphosphates to their deoxygenated counterparts, a key step in DNA synthesis. It is important to note that the expression of RRM2 but neither RRM1 nor RRM2b was consistently and significantly elevated in tamoxifenresistant cell line and xenograft samples as well as in publically available clinical data sets (Figure 4, E and F, and Supplementary Figure 7). In light of these findings and our observation that the ratio of the dNTP: NTP (deoxynucleotide triphosphate: nucleotide triphosphate) is higher in TAM-R in vitro and in vivo samples, we allude to the possibility that TAM resistance could be a reflection of increased RRM2 activity. However, other biochemical mechanisms, including a role for RRM1 and RRM2B, cannot be ruled out and need further examination. A recent report showed the induction of RRM2 by overexpressed AKT in TAM-R cells [70]. As AKT is known to promote proliferation and cell growth in multiple cancers, elevated expression of RRM2 could portray increased rate of DNA synthesis to support AKT-induced proliferation demand. Yet another possibility regarding role of RRM2 in the context of TAM resistance stems from our in silico findings (Supplementary Table 9) that suggest a potential regulation by estrogen receptor 1α (ESR1). Activating mutations of ESR1 have been reported by multiple groups to be associated with endocrine resistance [71,72]. These findings point to the possibility of increased RRM2 expression as being a downstream consequence of activated ESR1 in patients, a hypothesis that needs to be validated. In addition, a recent report in melanoma suggests that RRM2 could induce cellular senescence [73] and hence create a mechanism for the tumors to escape cytotoxic effect of the therapy. This is an interesting possibility that needs to be examined in the context of TAM resistance.
Importantly, a strong association between RRM2 expression and TAM resistance led us to test potential inhibitors whose activity has been reported to reverse RRM2 expression. Azacytidine, a well-known DNA methyltransferase inhibitor, was recently reported to decrease mRNA and protein levels of RRM2 in leukemic cell lines although its mechanism of action was not reported ( [60]). Consistent with their findings, RRM2 protein expression in our hands was significantly reduced on treatment of TAM-R cells with aza. In line with this, azatreated TAM-R cells showed significantly decreased rate of proliferation in the presence of TAM, indicative of potential resensitization to the TAM treatment. Although preliminary, these findings set the stage for additional experiments to determine the optimal concentration of aza required to supplement tamoxifen and to understand the mechanism(s) that drive this synergy.

Conclusions
Taken together, we have developed a novel bioinformatics method to integrate metabolomics and gene expression data from cancer cell lines and tissues to nominate key pathways that are altered and have prognostic value. RRM2, a key gene in pyrimidine metabolism, was found to be associated with aggressive breast tumors as well as TAM-R luminal BCas, and its pharmacological or genetic knockdown sensitized tumors to TAM. In summary, the study nominates RRM2 as a key marker for aggressive BCa including TAM-R tumors. In light of this finding, RRM2-associated metabolites could be developed as prognostic markers for BCa. In addition, the combination of aza with TAM could be explored in a preclinical setting to treat TAM-resistant BCas.