Introduction

In 2013, more than 200,000 women in the United States will be diagnosed with breast cancer (BRCa) [1]. Despite advances in treatment and earlier detection, nearly 40,000 women with BRCa will die from this disease [1]. In clinical practice, BRCa is divided into subgroups based on the expression of the estrogen receptor (ER), progesterone receptor (PR), and the status of gene amplification of human epidermal growth factor 2 receptor (HER2). The four predominant subtypes reported extensively in literature are categorized based on these receptors: luminal A (ER+ and/or PR+, HER2-), luminal B (ER+ and/or PR+, HER2+), HER2 (ER-, PR-, HER2+), and triple negative (ER-, PR-, HER2-). The luminal A and luminal B subtypes express ER or PR and have several treatment options due to their susceptibility to hormone-based adjuvant therapies that target these receptors, but the HER2 subtype, as its name implies, only has HER2 amplification which severely limits adjuvant treatment options [2]. Triple negative breast cancer (TNBC) lacks expression of ER and PR, and HER2 amplification and treatment is primarily limited to surgery and chemotherapy [3]. A subtype of TNBC is the basal or basal-like phenotype that is negative for all three receptors but expresses specific basal markers (cytokeratin 5/6, cytokeratin 14, cytokeratin 17, and epidermal growth factor receptor) [4]. This phenotype is more likely to undergo metastasis and is associated with poorer prognosis [5].

African American (AA) women have a higher mortality than Caucasian American (CA) women with BRCa [1]. Although socioeconomic status has been shown to be an independent predictor of mortality, the increase in mortality of AA women with BRCa cannot be explained through socioeconomic status alone [6-8]. AA women still suffered from higher mortality in studies and in a randomized clinical trial where AA and CA women had equal access to care [9-11]. These observations suggest a biological basis for the elevated mortality of AA women, and this hypothesis is supported by a study that shows AA women are more likely to develop tumors with worse pathological characteristics (e.g., larger tumor size, less differentiation of cancerous cells) [12]. Other studies have shown that AA breast cancer patients are more likely to have detrimental tumor subtypes such as ER- and PR-, triple negative, and basal-like breast tumors [13-18].

There is no proven cause of health disparity, but studies indicate that genetic differences between AA and CA tumors do exist. Martin et al. first performed genome-wide mRNA expression analysis of tumor epithelium from 18 AA and 17 CA patients [19]. They found over 400 differentially expressed genes and identified a two-gene signature, putative phosphoserine phosphatase-like protein (PSPHL) and beta-crystallin B2 (CRYBB2), which could distinguish between populations. Field et al. conducted a study with 26 pairs of matched AA and CA BRCa patients and showed that molecular profiles differ between AA and CA breast tumors despite being matched by pathological characteristics. They detected more than two dozen differentially expressed genes that are involved in cellular growth, differentiation, invasion, metastasis, and immune response [20]. Additionally, a study by Grunda et al. using 12 pairs of matched AA and CA BRCa patients assessed a set of 84 genes involved in breast carcinoma prognosis, response to therapy, estrogen signaling, and tumor aggressiveness of age- and stage-matched AA and CA tumor samples [21]. They identified 20 differentially expressed genes from this set, leading to their conclusion that gene expression differences may play a role in increased metastatic potential, resistance to therapy, and worse clinical outcome in AA women.

These previous reports are consistent with one exception: ribosomal protein L13 (RPL13) was lower in AA women in the Martin et al. study but higher in the Field et al. study [19,20]. This could be explained by the small sample size or possibly by variation in age, stage, or subtype between the study populations. Moreover, these studies did not analyze differentially expressed genes by stage or subtype, possibly due to lack of patient samples. Since AA women are more likely to be afflicted by worse subtypes it is apparent that comprehensive studies that include stage and subtype analyses as well as larger sample size are necessary to elucidate the genetic factors responsible for BRCa health disparity.

We used next generation sequencing (NGS) data from the Cancer Genome Atlas (TCGA) to determine differentially expressed genes and other transcripts between a large number of age- and stage-matched AA and CA patient primary tumor samples. We chose NGS data because of its accuracy and the ability to use it to determine differential expression between non-coding RNAs in addition to coding RNAs. This study represents, to our knowledge, the first time that NGS data was used in combination with a large patient cohort to investigate expression differences of genes and other transcripts between AA and CA BRCa patients, and to our knowledge, this work represents the largest and most comprehensive study on health disparity in AA BRCa to date. To our knowledge, we are the first to identify differentially expressed genes by stage and subtype between AA and CA tumors in addition to differentially expressed subnetworks and pathways.

There are several options to test for differential expression. The Poisson distribution and the negative binomial distribution are both discrete probability distributions that can model count data from a gene sequencer over a finite period of time, and many previous methods for differential expression analysis of NGS data have been based on the Poisson distribution [22,23]. However, the Poisson distribution often under-estimates the actual variations of expression data because it assumes the mean is equal to the variance [24,25]. The negative binomial distribution can tolerate instances of large biological variance between replicates and is often used to cope with the over-dispersion issue by allowing mean and variance to be different. With this in mind, we chose to use DESeq, an R Bioconductor package, for our analyses because it uses the negative binomial distribution to derive a test for differential expression [26,27].

Results

Table 1 shows the breakdown of patient demographics obtained from TCGA. AA women had nearly double the mortality of their age- and stage-matched CA counterparts with 18.87% mortality compared to 10.28% CA mortality, and this increase in mortality is despite patient samples being age- and stage-matched. As mentioned in the Introduction, this disparity is likely caused by both socioeconomic and biological factors. Information regarding socioeconomic status or access to care is not available at TCGA. Our observed mortalities are lower than previously reported population data for both races: 18.2% for CA women and 26.8% for AA women [1]. This is likely caused by comparably fewer patients with late stage breast cancer donating their tumor samples to TCGA.

Differential expression analysis at the individual gene level was firstly conducted to identify genes and other transcripts that differ between AA and CA BRCa patients using DESeq; secondly, pathway analysis and gene set enrichment analysis with the Pathway Interaction Database (PID) and Gene Set Enrichment Analysis (GSEA) was performed; thirdly, Gene Expression Network Analysis (GXNA) was used to identify differentially expressed subnetworks; finally, patients were stratified to study differences in stage- or subtype-specific gene expression again with DESeq (Figure 1).

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Figure 1. A flowchart summarizing the data analysis methodology.

https://doi.org/10.1371/journal.pone.0082460.g001

In total, 674 unique genes and other transcripts were shown to be differentially expressed between the various comparisons performed (Table S1). The number of unique genes was computed by combining gene names from the overall, stages I-III, luminal A, HER2, and triple negative comparisons into a single list. We then removed duplicate gene names from the list, and this new list without duplicates was then counted to give the total number of unique genes. To identify trends in gene expression, primary breast tumor expression data between AA and CA patients irrespective of subtype or stage (i.e., the “overall” comparison) was first compared. Three hundred forty-two genes and other transcripts, 1.7% of the 20,531 genes and other transcripts assessed, were identified by DESeq as differentially expressed between AA and CA primary breast tumors (P < 0.001, Table S2). One hundred ten of these genes and other transcripts showed increased expression in AA tumors while 232 were decreased. Thirty-seven of the 342 genes and other transcripts exhibited significant differential expression (i.e., log₂ fold-change greater than 1.0 or less than -1.0), and selected genes and other transcripts with high fold-change and relevance to breast cancer can be seen in Table 2.

The 342 differentially expressed genes and other transcripts from the overall comparison were searched against PID (Table 3). PID places genes into known pathways and ranks them by the probability that their associated genes are enriched in the input gene list, and a low P-value indicates a greater chance of enrichment. The top PID pathway was the tumor protein p73 (p73) transcription factor network (P = 4.75E-05). p73 is a tumor suppressor whose overexpression is seen in some aggressive breast cancer tumors and breast cancer cell lines [28]. The next most significant PID result was the aurora B signaling pathway (P = 1.07E-04). Aurora kinase B (AURKB) is a mitotic checkpoint kinase that mediates chromosome segregation and is overexpressed in a variety of cancers including lung, colorectal, and prostate [29-31]. The polo-like kinase (PLK1) signaling events pathway was also identified by PID (P = 2.35E-04). PLK1 is a kinase significantly overexpressed in TNBC that may be a potential therapeutic target for TNBC [32]. We saw differential expression in the BRCA1 associated RING domain 1 (BARD1) signaling events pathway (P = 4.70E-04). BARD1 interacts with tumor protein p53 (p53 or TP53); breast cancer 1, early onset (BRCA1); and breast cancer 2, early onset (BRCA2), all of which are important tumor suppressors in breast cancer [33-35]. The genes within the pathways identified by PID had almost unanimous increase in gene expression in AA tumors. The one exception was BARD1 signaling events pathway which had one gene, RAD50 homolog (S. cerevisiae) (RAD50), decrease in AA tumors. Additionally, we uploaded the genes from the Martin et al., Field et al., and Grunda et al. studies on health disparity to PID and compared the results with our own [19-21]. Out of all the previous works, our results only had the aurora B signaling pathway in common with the Martin et al. data [19].

GXNA was then used to identify differentially expressed subnetworks between AA and CA tumors. GXNA takes patient expression data as input and then outputs a ranked list of subnetworks that contain interactions involving the differentially expressed genes. GXNA identified two subnetworks, one involving p53 and another involving BRCA1, both with a majority of components upregulated in AA breast tumors (P < 0.001, Figure 2). STRING was used to visualize GXNA data and to show potential gene product interactions beyond what is shown in the GXNA interactions. Gene Set Enrichment Analysis did not identify any differentially expressed gene sets (P-value > 0.05 or with unacceptable FDR; results not shown).

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Figure 2. Differentially expressed subnetworks identified by Gene Expression Network Analysis.

Subnetworks containing p53 (A) and BRCA1 (B) were differentially expressed in AA tumors. Subnetworks were identified using GXNA and visualized using STRING. Starred results were not differentially expressed but were included in the subnetwork by GXNA. Values in parentheses are the mean fold changes of log₂-transformed AA expression relative to CA expression, calculated as log₂(CA)/log₂(AA). Gene names: HMGB2: BRCA1 - breast cancer 1, early onset; CCNB2 - cyclin B2; CDC25B - cell division cycle 25B; CDK1 - cyclin-dependent kinase 1; CKS2 - CDC28 protein kinase regulatory subunit 2; ELAVL1 - ELAV (embryonic lethal, abnormal vision, Drosophila)-like 1 (Hu antigen R); FANCA - Fanconi anemia, complementation group A; HMGB2 - high mobility group box 2; HSF1 - heat shock transcription factor 1; HSPA8 - heat shock 70kDa protein 8; PKMYT1 - protein kinase, membrane. associated tyrosine/threonine 1; PML - promyelocytic leukemia; PPP1R13L - protein phosphatase 1, regulatory subunit 13 like; PTMA - prothymosin, alpha; RAD50 - RAD50 homolog (S. cerevisiae); RAD51 - RAD51 recombinase; TP53 - tumor protein p53; TXN – thioredoxin.

https://doi.org/10.1371/journal.pone.0082460.g002

Stage-matched and then subtype-matched AA and CA tumors were next compared, but a lack of stage IV and luminal B AA tumors allowed us to only evaluate stages I through III and the luminal A, HER2, and triple-negative subtypes. Selected results from these comparisons are summarized in Table 4. Interestingly, very few genes and other transcripts overlapped between the stage comparisons (Figure 3). Twenty-six genes and other transcripts were differentially expressed in the stage I comparison (19 increased and 7 decreased in AA tumors, Table S3A), including BUB1 mitotic checkpoint serine/threonine kinase (BUB1; 1.05 log₂ fold-change; P = 9.80E-04) which is a potent prognostic factor for human breast cancer [36]. One hundred sixty-one genes were differentially expressed in the stage II comparison (134 increased and 27 decreased, Table S3B) including crystallin, beta B2 (CRYBB2; 2.00 log₂ fold-change; P = 3.27E-10) which was differentially expressed in AA breast, colorectal, and prostate cancers [19,20,37,38]. Two hundred twenty-three genes were differentially expressed in the stage III comparison (156 increased and 67 decreased, Table S3C), including estrogen receptor 1 (ESR1; -2.25 log₂ fold-change; P = 8.80E-05). Forty-six genes and other transcripts were differentially expressed in the luminal A comparison (39 increased and 7 decreased in AA tumors, Table S3D) including polycystin (PKD) family receptor for egg jelly (PKDREJ; -1.18 log₂ fold-change; P = 9.56E-04) which is point mutated in breast cancer [39]. Twenty-five genes and other transcripts were differentially expressed in the HER2 comparison (21 increased and 4 decreased, Table S3E) including TCRgamma alternative reading frame protein (TARP; 8.74 log₂ fold-change; P = 2.31E-04) which is a breast and prostate tumor-associated antigen [40]. Fifteen genes and other transcripts were differentially expressed in the triple negative comparison (10 genes increased and 5 decreased, Table S3F) including peptidoglycan recognition protein 1 (PGLYRP1; 2.99 log₂ fold-change; P = 7.64E-04) whose expression may reflect immune cell response to developing breast tumors [41].

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Figure 3. Venn diagram depicting overlap of differentially expressed genes and other transcripts between stage-matched African- and Caucasian-American tumors.

Increases or decreases indicate AA gene expression and are relative to CA expression. Gene names: FYCO1 - FYVE and coiled-coil domain containing 1; LOC90784 - Not Available; LOC285359 - Not Available; LRRC37A2 - leucine rich repeat containing 37, member A2; MEIS3P1 - Meis homeobox 3 pseudogene 1; NOTCH2NL - notch 2 N-terminal like; PRSS45 - protease, serine, 45.

https://doi.org/10.1371/journal.pone.0082460.g003

LOC90784, a transcript, was the only gene or transcript differentially expressed in more than one subtype (luminal A and triple negative). Additionally, it was differentially expressed in the overall, stage I, stage II, and stage III comparisons. This made it the only transcript or gene we found to be differentially expressed across six different comparisons. Interestingly, the expression of LOC90784 remained consistent across CA tumor stage and grade but was consistently lower in AA samples, and LOC90784 expression was inversely related to stage among AA patient samples and was the lowest in triple negative AA tumors (Figure 4).

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Figure 4. LOC90784, a long non-coding RNA, was differentially expressed across comparisons of African- and Caucasian-American tumors.

The expression of this transcript was consistent in CA tumors, but its expression was inversely related to stage and was lowest in AA with TNBC. P-values for the comparisons were overall: 1.46E-14, luminal A: 1.98E-04, stage I: 6.61E-04, stage II: 3.63E-08, stage III: 2.57E-05, and triple negative: 1.86E-09 (see Supplementary Tables). Error bars are standard error.

https://doi.org/10.1371/journal.pone.0082460.g004

These results share some agreement with previous studies (Table 5). Fifteen genes out of 424 were found to be in common with those reported by Martin et al. [19]. An additional two genes were in common with those identified by Martin et al. but the results are contradictory (deoxyuridine triphosphatase or DUT expression was higher in AA samples in our results but lower in the Martin et al. results; Zic family member 1 or ZIC1 was lower in AA samples in our results but higher in the Martin et al. results) [19]. Three out of 27 genes were found to be in common with those found by Field et al. (CRYBB2, mitochondrial ribosomal protein L48 or MRPL48, and zinc finger protein 395 or ZNF395) [20], and one gene out of twenty was shared with those found by Grunda et al. (ESR1) [21]. It is not straightforward to assess the statistical significance of the agreements between these studies and our own so we cannot rule out that the agreements of our studies are purely by chance.

Discussion

We identified 342 differentially expressed genes and other transcripts (P < 0.001) between AA and CA primary breast cancer tumors in the overall comparison. Thirty-seven of these exhibited significant change (i.e., log₂ fold-change > 1.0 or < -1.0), and selected breast cancer-associated genes are shown (Table 2). Several genes have increased expression in AA tumors, including a few with direct ties to breast cancer such as resistin (RETN). RETN is an adipocytokine whose expression level is significantly increased in AA tumors (2.25 log₂ fold-change; P = 3.05E-06). It is expressed in inflammatory cells in humans and plays a role in insulin resistance in mice [42,43]. Breast cancer patients show increased resistin concentrations over control [44], and a recent study indicates that high resistin expression in breast cancer tissue is associated with decreased survival and more malignant characteristics [45]. Its presence at such high expression levels suggests that resistin may play an important role in AA BRCa because of the strong link between obesity and cancer mortality [46-48]. Likewise, a number of genes have reduced expression in AA women, including ADAM metallopeptidase with thrombospondin type 1 motif, 15 (ADAMTS15; -1.29 log₂ fold-change; P = 1.82E-04). ADAMTS15 is a metalloprotease known to inhibit breast cancer cell migration [49], and it is possible that the reduced expression of this gene could have consequences in AA BRCa progression.

We noticed an increase in expression of CRYBB2 in AA samples (Table 2). This gene encodes a protein that is a major component of the vertebrate eye lens and whose mutation may cause cataracts [50]. Expression of this gene was increased in AA tumor samples [19], and its expression was increased in both nonmalignant and tumor samples [20]. Martin et al. used this gene to differentiate between AA and CA patients while Field et al. hypothesized that overexpression of this gene could possibly enhance AA tumorigenesis. Expression of CRYBB2 was also increased in a study comparing AA and CA colorectal cancer and in another study comparing AA and CA prostate cancer [37,38]. These last two results are intriguing since health disparity exists in both prostate cancer and colorectal cancer in the AA population. The fact that this gene is overexpressed in nonmalignant tissues could simply be a result of racial differences, but we only observed differential expression of CRYBB2 in our overall and stage II comparisons. We would expect to see CRYBB2 in more comparisons if this gene was always differentially expressed between races. We suggest further studies into the role, if any, which CRYBB2 plays in cancer and health disparity.

We saw elevated expression of all members of the aurora B signaling pathway identified by PID (P = 1.07E-04). Aurora B kinase (AURKB), a member of the pathway sharing its name, had nearly doubled expression in AA compared to CA (0.89 log₂ fold-change; P = 3.49E-05; Table S2). AURKB is a mitotic checkpoint kinase that mediates chromosome segregation and is overexpressed in a variety of cancers including lung, colorectal, and prostate [29-31], and it interacts with the aforementioned BARD1 pathway [35]. There has been no evidence that AURKB is overexpressed in breast cancer, but aurora kinase family inhibitors have been suggested as treatments for cancer. Breast cancer cell lines treated with the highly selective AURKB inhibitor Barasertib show anti-neoplastic effects [51-53]. Based on the increased expression of AURKB and other aurora B signaling pathway members in AA tumors, we speculate that this drug may be used as an effective treatment for African American breast cancer patients.

We used GXNA to identify differentially expressed subnetworks in AA BRCa. Subnetworks differ from pathway analysis results because they consist of genes that share interactions with one-another across multiple pathways, and this allows for the identification of differentially expressed subnetworks that are not limited to a single pathway. We identified two differentially expressed subnetworks related to BRCa with GXNA, one centered on p53 (P = 0.001) while the other involved BRCA1 (P = 0.001, Figure 2). Both of these pathways had a majority of genes with increased expression in AA samples. p53 is well-studied tumor suppressor that can activate DNA repair as well as initiate apoptosis [54], and p53 status is also an independent predictor of survival in AA women with BRCa [55]. Although p53 was not differentially expressed in our results, many of its signaling neighbors were increased. BRCA1 is an important DNA repair enzyme whose loss is associated with dramatically increased BRCa incidence [56], and BRCA1 and BRCA2 mutations are seen in high-risk AA women with BRCa [57]. Like p53, BRCA1 was not differentially expressed in our results but many of its signaling neighbors were increased. The GXNA results are strengthened by our pathway analysis result that showed increased activity in the BARD1 pathway whose members interact with both p53 and BRCA1 [33-35]. These results strongly suggest increased activity in the p53 and BRCA1 subnetworks in AA women, but these results are somewhat contradictory: higher expression or activity in two pathways related to tumor suppression and DNA repair should logically have a beneficial effect and should not contribute to health disparity. However, we suggest that there may be dysregulation up or downstream of these pathways as a result of a mutation, and the increase in activity of these pathways could be explained by the patient’s cells attempting to compensate for dysregulation. p53-expressing tumors are more frequent in AA women with TNBC when compared to their CA counterparts with TNBC [58], so the increase in expression of the p53 subnetwork could be partially explained by this fact.

Our stage III comparison showed that AA tumors had significantly lower ESR1 (-2.25 log₂ fold-change; P = 8.80E-05; Table 4). This comparison was our only comparison where ESR1 was differentially expressed between AA and CA samples, and Grunda et al. saw a similar decrease in expression of ESR1 in their study [21]. We believe this finding suggests that AA women may have reduced ER expression in later stages or that AA women may be more susceptible to mutations causing the ER- phenotype.

LOC90784, a transcript, was the only gene or transcript differentially expressed in the overall, stage I, stage II, stage III, luminal A, and triple negative comparisons. The expression of LOC90784 was very similar across the CA population regardless of stage or subtype, but its expression was inversely related to stage among AA samples and was lowest in AA women with TNBC (Figure 4). Little is known about this transcript other than it is found on chromosome 2 in humans (2p11.2), is 3,653 bases long, shares 100% sequence with polymerase (RNA) I polypeptide A, 194kDa (POLR1A), and produces long, non-coding RNA (lncRNA) of undetermined function (NCBI Gene ID: 90784, updated on 16-Aug-2013). Non-coding RNA do not encode for protein, yet are alternatively spliced and processed into smaller products [59]. A search of miRBase (accessed March 13^th, 2013) revealed that LOC90784 is not processed into any known microRNA [60]. However, lncRNAs are expressed in a disease-specific manner that makes them potential therapeutic targets since they are implicated in BRCa and promoting metastasis [61-63]. Another possibility is that LOC90784 is a miscategorized, alternatively spliced variant of POLR1A, but we have no evidence of this and further experiments are needed.

One emerging trend was that 26 genes and other transcripts were identified as differentially expressed in stage I, then 161 in stage II, and then 223 in stage III. This suggests that stage I tumors are more genetically similar to one another and that similarity is lost in higher stages. This increase in differentially expressed genes may be due to the increased rate of changes over time in a dysfunctional, cancerous cell due to genomic instability [64], but this finding may also suggest that AA women with BRCa have a higher rate of mutation than their CA counterparts. It would have been interesting to observe if this trend was maintained in stage IV tumors, but no stage IV AA data was available for analysis at the time of writing.

This study further provides molecular insights on breast cancer health disparity and identifies several genes, other transcripts, and pathway targets for additional investigation. We observed increases in differentially expressed genes in later stages of cancer, suggesting higher genomic instability in African American patients. Several genes were differentially expressed including resistin, an adipocytokine involved in insulin resistance, obesity, and breast cancer, which had over four times higher expression in AA patients. A long, non-coding RNA, LOC90784, which may have protective activity in breast cancer, was significantly reduced in AA patients and was the lowest in TNBC. There was also higher expression in a number of signaling pathways related to cancer, such as aurora B, BRCA1, and p53, which suggests up- or down-stream dysregulation within these signal transduction pathways.

Methods

Supporting Information

Acknowledgments

We acknowledge the Florida State University shared High-Performance Computing facility and staff for contributions to results presented in this paper. This work was in part supported by Elsa U. Pardee Foundation to Dr. Sang and the Florida State University to Dr. Sang and Dr. Zhang. We appreciate the generous support of anonymous donors for the Endowed Professorship in Cancer Research to Dr. Sang.