Abstract

   To investigate the biologic relevance and clinical implication of genes
   involved in multiple gene expression signatures for breast cancer
   prognosis, we identified 16 published gene expression signatures, and
   selected two genes, MAD2L1 and BUB1. These genes appeared in 5
   signatures and were involved in cell-cycle regulation. We analyzed the
   expression of these genes in relation to tumor features and disease
   outcomes. In vitro experiments were also performed in two breast cancer
   cell lines, MDA-MB-231 and MDA-MB-468, to assess cell proliferation,
   migration and invasion after knocking down the expression of these
   genes. High expression of these genes was found to be associated with
   aggressive tumors and poor disease-free survival of 203 breast cancer
   patients in our study, and the association with survival was confirmed
   in an online database consisting of 914 patients. In vitro experiments
   demonstrated that lowering the expression of these genes by siRNAs
   reduced tumor cell growth and inhibited cell migration and invasion.
   Our investigation suggests that MAD2L1 and BUB1 may play important
   roles in breast cancer progression, and measuring the expression of
   these genes may assist the prediction of breast cancer prognosis.

Introduction

   Accurately predicting the prognosis of breast cancer remains a
   significant challenge [[41]1]. Many clinical, pathologic and molecular
   markers have been identified for breast cancer prognosis, including
   disease stage, tumor grade and histology, lymph node involvement,
   hormone/growth factor receptor status and recently molecular subtypes,
   but none of them provides ideal accuracy in predicting prognosis and
   treatment response. High-throughput analyses, based on microarray chips
   and other technologies, are used to determine gene expression
   “signatures” for improved accuracy of prognosis. The first such
   signature was reported in 2002, which was on based on the expression of
   70 genes using a DNA-microarray technology [[42]2]. Since then, more
   than a dozen of gene expression signatures have been published for
   breast cancer prognosis [[43]3–[44]15]. Interestingly, when comparing
   the genes in the signatures, only a small number are shared by these
   signatures, and most of the genes are not overlapping. Reasons for this
   disparity in gene lists are probably multiple, including diverse
   patient populations, disease characteristics, analytical technologies,
   tissue preservation and preparation methods, and some degree of false
   positivity. Because of the inconsistency of gene inclusion across
   signatures as well as the feasibility and cost of using such signatures
   in patient management, many of the expression signatures have not been
   confirmed in prospective studies. To date, only two prognostic
   signatures [[45]2, [46]16] are being evaluated in clinical trials for
   utility [[47]17, [48]18], but the results will not be available until
   2020.

   While many studies are still aiming to develop new methods and
   techniques to improve the accuracy of prediction with the use of gene
   signatures, the biological relevance or pathologic involvement of the
   genes in signatures are often overlooked. Usually, the genes in a
   signature involve a number of biological processes and functions,
   making it difficult to identify key components that may drive tumor
   progression. For example, the first 70-gene signature [[49]2] contains
   genes involved in multiple biological activities, including cell cycle
   regulation, cell invasion and metastasis, angiogenesis, and signal
   transduction. Which process play key roles in tumor progression is
   unclear. Furthermore, the effects on tumor cells of many genes in the
   signatures are unknown. Thus, in an attempt to address some of these
   issues, we identified and compared genes from 16 publications that have
   reported gene-expression signatures for breast cancer prognosis. From
   the genes that appeared in multiple expression signatures, we selected
   two, MAD2L1 (Mitotic Arrest Deficient 2-like 1) and BUB1 (Budding
   Uninhibited by Benzimidazoles 1), for further validation of their
   associations with patient survival in our own and other studies, as
   well as for in vitro evaluation of their biological involvements in
   breast cancer.

Material and Methods

Selection of gene expression signatures

   We searched the PubMed database (US National Library of Medicine) using
   the phrases “breast cancer”, “gene expression”, “signature”, and
   “survival”. The search was updated in May 2015. The studies selected
   for our analysis were based on the following conditions: a) it was an
   initial report of a gene signature associated with breast cancer
   survival; b) an entire list of genes involved in the signature was
   reported; and c) gene expression data were generated from microarray
   analysis. Following the criteria, a total of 14 studies (or signatures)
   were selected [[50]2–[51]15, [52]18, [53]19]. Two additional studies by
   van Vliet et al. [[54]20] and Dai et al. [[55]21] were also included in
   our analysis. These studies analyzed the same data from Van’s Veer
   [[56]2], but used very different strategies and generated different
   signatures with regard to the lists of genes. Therefore, they were
   considered as independent studies. The study conducted by Van’s Veer et
   al [[57]2] identified a 231-gene signature in the initial stage. We
   used the genes in the initial signature for our analysis. A well-known
   EndoPredict signature based on RT-qPCR analysis was also included in
   our study [[58]12].

Breast cancer patients

   We recruited 203 female patients diagnosed with primary breast cancer
   from the University Hospital at University of Turin between January
   1998 and July 1999. The patients were enrolled in the study before
   surgery and were followed through February 2007. The study was approved
   by the ethical review committee of University Hospital at University of
   Turin. All participants provided written informed consent. Fresh breast
   tumor samples removed during surgery were snap-frozen in liquid
   nitrogen immediately after resection and stored at -80°C until
   analysis. Clinical and pathological characteristics of the patients are
   shown in the result section.

RNA extraction and analysis

   Protocols for RNA extraction and gene expression analysis have been
   described elsewhere [[59]22]. Briefly, total RNA was extracted from
   fresh-frozen tumor samples according to the conventional
   phenol-chloroform method following manual pulverization of tissue
   specimens in liquid nitrogen. The RNA samples were purified and
   concentrated by RNeasy MinElute Cleanup Kit (Qiagen Inc., Valencia,
   CA). Concentrations and integrities of total RNA were analyzed using
   the RNA 6000 Nano LabChip Kit and Agilent 2100 Bioanalyzer. Microarray
   analyses were performed using the Illumina Expression BeadChip
   (HumanRef-8 v3) following the manufacturer’s protocol.

Antibodies

   Antibodies used for western blot analysis were purchased from various
   companies. Anti-MAD2L1 (D8A7, #4636) antibody and horseradish
   peroxidase (HRP)-conjugated secondary antibody were purchased from Cell
   Signaling Technology (Danvers, MA), anti-BUB1 antibody (ab54893) was
   from Abcam (Cambridge, MA), and anti-β-actin antibody (A2228) was from
   Sigma-Aldrich (St. Louis, MO).

Cell lines and cultures

   Human breast cancer cell lines MDA-MB-231 (MB231) and MDA-MB-468
   (MB468) used for our in vitro experiments were supplied by Dr. Richard
   Yip at University of Hawaii Cancer Center, who obtained them from NCI
   as part of the NCI-60 DTP Human Tumor Cell Screening Panel. The cells
   were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented
   with 10% fetal bovine serum (FBS) and 100 units/ml of
   penicillin/streptomycin (Pen/Strep) at 37°C in a humidified atmosphere
   of 5% CO[2].

Western blots

   In siRNA knockdown experiments, protein expression was analyzed by
   western blot. Cells were treated with a lysis buffer [1% Triton x-100,
   150 mM NaCl, 50 mM Tris-Cl, pH 7.4, 0.5 mM EDTA, 1 mM sodium
   orthovanadate, 5 mM beta glycerol phosphate, and 1x protease inhibitor
   cocktail (Roche, Indianapolis, IN)] after transfection with indicated
   siRNAs for 36 h, and protein concentration was measured. Proteins
   (40–60 μg) from the cell lysates were separated by SDS-PAGE (10% sodium
   dodecyl sulfate-polyacrylamide gel electrophoresis) under denaturing
   conditions, and then transferred to polyvinylidene difluoride (PVDF)
   membranes (Millipore, Billerica, MA). After transferring, the membranes
   were blocked with 5% non-fat milk for 45 minutes, and then incubated
   with a primary antibody followed by incubation with a secondary
   antibody. The signals were detected by the enhanced chemiluminescence
   system (ECL) as described by the manufacturer (Pierce, Rockford, IL).

siRNA transfection

   The ON-TARGET plus SMARTpool siRNA of human MAD2L1 and BUB1 and
   negative control ON-TARGET plus Non-targeting Pool were purchased from
   Dharmacon (Lafayette, CO). The sequences of MAD2L1 and BUB1 siRNAs as
   well as control siRNAs are (a) ON-TARGET plus SMARTpool Human MAD2L1
   siRNA: UUACUCGAGGCAGAAAUA (siMAD2L1-1), CUACUGAUCUUGAGCUCAU
   (siMAD2L1-2), GGUUGUAGUUAUCUCAAAU (siMAD2L1-3) and GAAAUCCGUUCAGUGAUCA
   (siMAD2L1-4); (b) ON-TARGET plus SMARTpool Human BUB1 siRNA:
   CGAAGAGUGAUCACGAUUU (siBUB1-1), CAAAGAAGGGUGUGAAACA (siBUB1-2),
   GAAUGUAAGCGUUCACGAA (siBUB1-3), and GCAACAAACCAUGGAACUA (siBUB1-4); and
   (c) ON-TARGET plus Non-targeting siRNA: UGGUUUACAUGUCGACUAA (siRNA-1),
   UGGUUUACAUGUUGUGUGA (siRNA-2), UGGUUUACAUGUUUUCUGA (siRNA-3), and
   UGGUUUACAUGUUUUCCAU (siRNA-4). Cells were seeded onto 6-well plates in
   antibiotic-free medium plus 10% FBS overnight. When 70% confluent,
   cells were transfected with Lipofectamine RNAiMAX (Invitrogen)
   according to the manufacturer’s protocol. The final concentration of
   siRNAs was 50 nM. After transfection, cells were cultured for 36 hours,
   and then analyzed by western blot for protein expression of MAD2L1 and
   BUB1, as well as cell proliferation, in vitro migration and invasion
   assays.

Cell proliferation assay

   Cells were plated onto 96-well plates at a density of 3 x 10^3 cells
   per well, and cultured in the complete medium at 37°C under a
   humidified atmosphere containing 5% CO[2]. At 0, 24, 48, and 72 hours
   of incubation, cell proliferation reagent WST-1 was added into the
   wells in accordance with the manufacturer’s instructions (Roche,
   Penzberg, Germany). Cells were continuously incubated for 2 hours with
   WST-1, and then the color in each well of the plate was measured by a
   microplate spectrophotometer (Biotek Synergy 2, Winooski, VT) at the
   450 nm wavelength. The light absorbance was proportional to the numbers
   of cells in each well. The results of measurement at different time
   points of incubation were referenced to the measurement at 0 hours of
   incubation. Each proliferation assay was performed in triplicate.

Transwell migration and invasion assays

   In vitro cell migration and invasion assays were performed in 24-well
   plates with the Costar Transwell permeable membrane support with 8.0-μm
   pore size (Corning, NY). In the cell invasion assay, 200 μl control and
   siRNA knockdown cells in serum-free medium (1 x 10^4 cells per well)
   were seeded in the upper chambers coated with growth-factor-reduced
   Matrigel, 1 mg/ml (BD Pharmingen, San Diego, CA). The lower chambers
   were filled with 600 μl culture medium containing 10% FBS. Cell
   migration assays were performed similarly but without Matrigel coating.
   Cells invading or migrating to the underside of the filter membrane
   were stained with HEME 3 Solution (Fisher Diagnostics, Middletown, VA)
   after 36 hours of culture, and then counted using an Olympus CKX41
   microscope with an Infinity 2 camera. All experiments were repeated
   three times with triplicate wells.

Statistical analysis

   DAVID Bioinformatics Resources v6.7 ([60]http://david.abcc.ncifcrf.gov)
   was used for the pathway enrichment analysis, with recommended standard
   parameters selected in the analysis [[61]23, [62]24]. KEGG (Kyoto
   Encyclopedia of Genes and Genomes) database
   ([63]http://www.genome.ad.jp/kegg/) was employed for the analysis of
   protein functions in different pathways. All the microarray data were
   pre-processed by the BeadStudio Software and analyzed using the R
   statistical software and Bioconductor [[64]25]. The Lumi R package was
   used to normalize the data [[65]26]. Wilcoxon rank-sum test was
   performed to assess the association of gene expression and
   clinicopathologic characteristics of breast cancer patients.
   Kaplan-Meier survival analysis and Cox proportional hazards regression
   models were used to analyze the association of gene expression with
   breast cancer survival. Log-rank test was performed for Kaplan-Meier
   survival curve comparisons. Hazards ratios (HRs) and 95% confidence
   intervals (CIs) were calculated in the Cox regression analysis, and
   both univariate and multivariate models were developed. In multivariate
   analysis, HRs were adjusted for patient age at surgery, tumor grade,
   disease stage, and estrogen receptor (ER) and progesterone receptor
   (PR) status. Two types of survival outcomes were considered in survival
   analysis, overall survival (OS) defined as the time between the date of
   surgery and date of death or last follow-up, and relapse-free survival
   (RFS) defined as the period from surgery to recurrence or last
   follow-up. MAD2L1 and BUB1 expression were categorized into low, medium
   and high based on their tertile distributions. To validate the
   associations of MAD2L1 and BUB1 expression with breast cancer survival,
   an online tool named Gene expression-based Outcome for Breast cancer
   Online (GOBO) ([66]http://co.bmc.lu.se/gobo) was used [[67]27]. All of
   the values presented in the figures of in vitro experiments are means
   and standard deviations. Two-tailed Student t test was performed to
   determine the differences in means between groups, and P<0.05 was
   considered statistically significant. All statistical analyses were
   performed using the Statistical Analysis System, version 9.2, (SAS
   Institute Inc., Cary, NC) and R software (version 3.0.2).

Results

Genes in 16 reported expression signatures

   Studies reported gene expression signatures in association with breast
   cancer prognosis are listed in [68]S1 Table. These signatures included
   a total of 1,399 distinct genes. Of them, 1,138 were found to have
   official names. One gene, BIRC5 (also known as Survivin), was included
   in 7 signature lists, another, MYBL2, was in 6 lists, 5 genes (BUB1,
   CENPF, MAD2L1, RRPM2, PRC1) were in 5 lists, and 12 genes were in 4
   lists. The complete list of genes appearing in 3 or more signature
   lists is provided in [69]S2 Table. Using the DAVID Bioinformatics
   Resources v6.7 to analyze the 1,138 genes for pathway enrichment, we
   found four significantly enriched pathways. Cell cycle was the most
   significant one (Benjamini-Hochberg corrected p = 10^−9.5), followed by
   p53 signaling pathway (p = 10^−4.2), pathways in cancer (p = 10^−3.6),
   and DNA replication (p = 0.016). We found that three genes involved in
   multiple signatures were also associated with survival outcomes in our
   study, including MAD2 mitotic arrest deficient-like 1 (MAD2L1),
   pituitary tumor-transforming gene-1 (PTTG1), and budding uninhibited by
   benzimidazole 1 (BUB1). All of these genes were involved in the cell
   cycle pathway. Since the effects of PTTG1 on breast cancer cells have
   been studied previously by Yoon at el. [[70]28], we focused our
   analyses on MAD2L1 and BUB1.

Associations of MAD2L1 and BUB1 expression with clinical and pathologic
features

   Using the gene expression data from our microarray analysis of breast
   cancer [[71]22], we examined the associations of MAD2L1 and BUB1
   expression with tumor features and disease outcomes. [72]Table 1 shows
   the clinical and pathologic characteristics of breast cancer patients
   in association with MAD2L1 and BUB1 expression. Patients with PR
   negative, ER negative, and high grade tumors had higher expression of
   MAD2L1 and BUB1 compared to those with PR positive, ER positive, and
   low grade tumors, respectively. Patient age, disease stage and lymph
   node involvement were not associated with MAD2L1 or BUB1 expression.

Table 1. Associations of MAD2L1 and BUB1 expression with clinical and
molecular characteristics of breast cancer.

   Variables MAD2L1 BUB1
   Low No. (%) Middle No. (%) High No. (%) P Low No. (%) Middle No. (%)
   High No. (%) P
   Age at surgery 0.375 0.113
   ≤57.11 35(34.31) 30(29.42) 37(36.27) 37(36.27) 27(26.48 38(37.25)
   >57.11 33(32.35) 39(38.24) 30(29.41) 31(30.39) 41(40.20) 30(29.41)
   Estrogen receptor 0.027 0.00036
   Positive 47(37.01) 47(37.01) 33(25.98) 52(40.94) 45(35.43) 30(23.62)
   Negative 21(27.27) 22(28.57) 34(44.16) 16(20.78) 23(29.87) 38(49.35)
   Progesterone receptor 0.012 0.000002
   Positive 42(41.18) 36(35.29) 24(23.53) 49(48.04) 34(33.33) 19(18.63)
   Negative 25(25.51) 32(32.65) 41(41.84) 18(18.37) 33(33.67) 47(47.96)
   Lymph node status 0.254 0.662
   Positive 31(31.31) 39(39.39) 29(29.29) 30(30.30) 35(35.35) 34(34.34)
   Negative 37(35.24) 30(28.57) 38(36.19) 38(36.19) 33(31.43) 34(32.38)
   Grade 0.000001 < .000001
   1 18(69.23) 4(15.38) 4(15.38) 15(57.69) 7(26.92) 4(15.38)
   2 29(37.18) 33(42.31) 16(20.51) 38(48.72) 25(32.05) 15(19.23)
   3 20(20.41) 31(31.63) 47(47.96) 13(13.27) 36(36.73) 49(50.00)
   Disease stage 0.237 0.093
   I 28(42.42) 22(33.33) 16(24.24) 26(39.39) 26(39.39) 14(21.21)
   II 34(30.63) 36(32.43) 41(36.94) 36(32.43) 34(30.63) 41(36.94)
   III, IV 6(22.22) 11(40.74) 10(37.04) 6(22.22) 8(29.63) 13(48.15)
   [73]Open in a new tab

   To evaluate whether MAD2L1 and BUB1 are over-expressed in breast
   cancer, a dataset ([74]GSE37751) was downloaded from the Gene
   Expression Omnibus (GEO) website ([75]www.ncbi.nlm.nih.gov/geo). This
   dataset included gene expression data from 61 breast cancer and 47
   adjacent non-tumor tissues analyzed by the GeneChip Human Gene 1.0 ST
   arrays (Affymetrix, Santa Clara, CA). The expression data were
   processed by the RMA algorithm using the Affymetrix Expression Console
   software [[76]29]. Both MAD2L1 and BUB1 expression were found to be
   higher in tumor than in adjacent non-tumor tissues (5.97±0.66 versus
   5.36±0.38 for MAD2L1, 6.80±1.14 versus 5.50±0.74 for BUB1,
   respectively, and both P<0.0001) in this database. Similar differences
   were also observed in another GEO dataset ([77]GSE29044) [[78]30].

Associations of MAD2L1 and BUB1 expression with overall and relapse-free
survival

   We further investigated the associations of MAD2L1 and BUB1 expression
   with breast cancer survival. [79]Fig 1 shows that high expression of
   MAD2L1 was significantly associated with increased risk of disease
   recurrence and death. Patients with high tumor expression of BUB1 also
   had poorer relapse-free and overall survival compared to those with low
   tumor expression. The associations of MAD2L1 and BUB1 expression with
   relapse-free survival remained statistically significant after
   adjusting for age at surgery, ER and PR status, tumor grade, and
   disease stage ([80]Table 2). The relationships of breast cancer
   survival with MAD2L1 and BUB1 expression were also analyzed in the GOBO
   database consisting of 914 tumor samples from 11 publically available
   microarray datasets. The results of the GOBO database analysis were
   similar to the findings of our study. High expression of MAD2L1 and
   BUB1 were significantly associated with poor relapse-free survival
   ([81]Fig 2B and 2D). High MAD2L1 expression was also associated with
   poor overall survival ([82]Fig 2A).

Fig 1. Kaplan-Meier survival curves by different levels of MAD2L1 and BUB1
expression in 203 breast cancer patients.

   [83]Fig 1
   [84]Open in a new tab

   A) Overall survival (OS) by low, medium and high MAD2L1 expression; B)
   Relapse-free survival (RFS) by low, medium and high MAD2L1 expression;
   C) Overall survival (OS) by low, medium and high BUB1 expression; (D)
   Relapse-free survival (RFS) by low, medium and high BUB1 expression.

Table 2. Associations of MAD2 and BUB1 expression with overall and
relapse-free survival.

   Variables Unadjusted Adjusted[85]^*
   Overall Relapse-free Overall Relapse-free
   HR 95% CI HR 95% CI HR 95% CI HR 95% CI
   MAD2L1
   low 1 1 1 1
   Mid 0.786 0.326 1.896 1.209 0.581 2.514 0.572 0.231 1.417 0.877 0.41
   1.876
   High 2.163 1.042 4.487 3.177 1.671 6.042 1.528 0.673 3.472 2.08 1.027
   4.214
   P for trend 0.0257 0.00016 0.223 0.0183
   BUB1
   low 1 1 1 1
   Mid 1.269 0.501 3.216 2.067 0.996 4.289 1.214 0.439 3.355 1.652 0.744
   3.67
   High 3.263 1.459 7.298 3.378 1.691 6.746 2.543 0.96 6.734 2.329 1.037
   5.231
   P for trend 0.0019 0.00038 0.033 0.0054
   [86]Open in a new tab

   * Adjusted for age at surgery, tumor grade, disease stage, ER status,
   and PR status.

Fig 2. Kaplan-Meier survival curves by different levels of MAD2L1 and BUB1
expression in the GOBO database.

   [87]Fig 2
   [88]Open in a new tab

   A) Overall survival (OS) by low, medium and high MAD2L1 expression; B)
   Relapse-free survival (RFS) by low, medium and high MAD2L1 expression;
   C) Overall survival (OS) by low, medium and high BUB1 expression; (D)
   Relapse-free survival (RFS) by low, medium and high BUB1 expression.

Suppression of MAD2L1 and BUB1 expression in breast cancer cell lines

   To evaluate the biological effects of MAD2L1 and BUB1 on breast cancer,
   we designed two RNA interference (RNAi) assays to knockdown the
   expression of MAD2L1 or BUB1. The experiments were performed in two
   triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468,
   which had high expression of endogenous MAD2L1 and BUB1. Cell growth,
   invasion and migration were evaluated in our in vitro experiments.
   Western blot analyses showed that MAD2L1 and BUB1 proteins were
   significantly declined after the cells were transfected with siRNAs
   specific for MAD2L1 or BUB1 (Figs [89]3 and [90]4). Compared with those
   transfected with scrambled siRNA, cells with MAD2L1 or BUB1 siRNA had
   lower cell proliferation. Cell invasion and migration were also reduced
   in those treated with MAD2L1 or BUB1 siRNA. Taken together, these
   results suggest that low MAD2L1 or BUB1 expression may inhibit breast
   cancer cell proliferation, invasion, and migration.

Fig 3. Effects of MAD2L1 or BUB1 depletion on the phenotype of MDA-MB-231
cancer cells in vitro.

   [91]Fig 3
   [92]Open in a new tab

   A) MAD2L1 protein expression by western blot analysis, β-actin used as
   control. B) Knockdown of MAD2L1 expression inhibited cell
   proliferation. C) Knockdown of MAD2L1 expression inhibited cell
   migration. D) Knockdown of MAD2L1 expression inhibited cell invasion.
   E) Representative pictures of cell migration and invasion before and
   after knockdown. F) BUB1 protein expression by western blot analysis,
   β-actin used as control. G) Knockdown of BUB1 expression inhibited cell
   proliferation. H) Knockdown of BUB1 expression inhibited cell
   migration. I) Knockdown of BUB1 expression inhibited cell invasion. J)
   Representative pictures of cell migration and invasion before and after
   knockdown. * = P<0.001.

Fig 4. Effects of MAD2L1 or BUB1 depletion on the phenotype of MDA-MB-468
cancer cells in vitro.

   [93]Fig 4
   [94]Open in a new tab

   A) MAD2L1 protein expression by western blot analysis, β-actin used as
   control. B) Knockdown of MAD2L1 expression inhibited cell
   proliferation. C) Knockdown of MAD2L1 expression inhibited cell
   migration. D) Knockdown of MAD2L1 expression inhibited cell invasion.
   E) Representative pictures of cell migration and invasion before and
   after knockdown. F) BUB1 protein expression by western blot analysis,
   β-actin used as control. G) Knockdown of BUB1 expression inhibited cell
   proliferation. H) Knockdown of BUB1 expression inhibited cell
   migration. I) Knockdown of BUB1 expression inhibited cell invasion. J)
   Representative pictures of cell migration and invasion before and after
   knockdown. * = P<0.001.

Discussion

   More than a dozen gene expression signatures involving over 1,300 genes
   have been published for breast cancer prognosis. Among them, no single
   gene was included in all of the signatures, and only two genes (MYBL2,
   BIRC5) were shared by six or seven signatures. These genes were
   reported to be associated with breast cancer survival, and suppressing
   the expression of these genes inhibited tumor cell proliferation
   [[95]31, [96]32]. Five genes appeared in five signatures, and twelve
   genes in four signatures. This low frequency of genes overlapping in
   the signatures suggests that the reported expression signatures are
   highly heterogeneous. This heterogeneity most likely reflects a
   diversity of patient populations or tumor specimens with regard to
   their clinical and pathologic characteristics. It is also possible that
   some of the results were observed by chance or from false-positive
   findings. To exclude this possibility, validation of the signature
   genes in independent studies or distinct patient populations is
   necessary.

   In our study of microarray expression data from 203 breast cancer
   patients, we analyzed the genes present in multiple signatures, and
   found three genes (MAD2L1, PTTG1 and BUB1) significantly associated
   with disease-free or overall survival. Of these genes, PTTG1 has been
   investigated by Yoon et al. who demonstrated the possible biological
   relevance of the gene to breast cancer [[97]28]. No reports were found
   for MAD2L1 and BUB1. Based on the pathway enrichment analysis of the
   genes involved in multiple expression signatures for prognosis, the
   cell cycle pathway was suggested to be the most significantly enriched.
   All the three genes mentioned above are in this pathway.

   Genes included in the expression signatures can be involved in multiple
   biological functions and cellular activities. In the studies exploring
   gene expression profiles as signatures for survival outcomes,
   biological and pathologic relevance of the genes to cancer in the
   signatures are often not evaluated experimentally and validated
   independently [[98]33]. Breast cancer is a heterogeneous disease
   characterized by distinct pathologic features, disparate treatment
   responses, and varied disease outcomes [[99]34]. These differences may
   reflect specific gene expression signatures. Using the genes in
   signatures, our pathway enrichment analysis showed that cell cycle is
   the most significant pathway. As cell cycle involves the processes of
   cell growth and division, and uncontrolled cell proliferation is a
   hallmark of cancer, [[100]35, [101]36], it is not difficult to imagine
   why these signatures are predictive of disease prognosis. Thus, in this
   study, we focused on MAD2L1 and BUB1, two critical mitotic checkpoint
   genes which play an important role in the mitotic process. Our
   investigation showed that high MAD2L1 or BUB1 expression was associated
   with poor prognosis of breast cancer, and moreover in vitro suppression
   of their expression resulted in reduced cell proliferation and less
   aggressive cell behavior. In addition, we found from publicly available
   data that expression of MAD2L1 and BUB1 was higher in tumor than in
   adjacent non-tumor tissues. Collectively, all of these observations
   suggest that MAD2L1 and BUB1, two genes present in multiple gene
   expression signatures for breast cancer prognosis, may be important
   molecules influencing tumor cell activity and patient survival.

   Under normal physiologic circumstances, cell cycle is tightly
   controlled by several mechanisms that regulate cell division and
   proliferation. One of the primary cell-cycle control mechanisms is the
   mitotic checkpoints in mitosis [[102]37]. As part of the mitotic
   checkpoints, the MAD (mitotic arrest-deficient) and BUB (budding
   uninhibited by benzimidazole) gene families are essential components of
   the spindle checkpoint, and were first identified by genetic screening
   of budding yeast for mutants [[103]38]. MAD2L1 is required in mitosis
   when chromosomes are unattached to the mitotic spindle that maintains
   chromosomal segregation, and is involved in the spindle checkpoint
   during mitosis [[104]39]. Dysregulation of these genes are associated
   with chromosomal instability and substantial aneuploidy which occur
   often in cancer [[105]40, [106]41]. One study revealed that mRNA levels
   of many spindle checkpoint genes (MAD1L1, MAD2L1, MAD2L2, BUB1, BUB1B,
   BUB3, CDC20 and TTK) were almost uniformly increased in breast cancer
   cell lines relative to the levels in normal breast cells (MCF10A and
   primary mammary gland); high expression was also observed in high-grade
   primary breast cancer [[107]42]. In our study, both MAD2L1 and BUB1
   were expressed substantially higher in high-grade than in low-grade
   tumors. Our analysis of MAD2L1 and BUB1 in two datasets ([108]GSE37751
   and [109]GSE29044) also demonstrated higher expression in breast cancer
   than in adjacent non-tumor tissues.

   One review has pointed out that high levels of MAD2L1 or BUB1 could
   lead to the formation of aggressive tumors in multiple organs
   [[110]43]. MAD2L1 has been found to be overexpressed in several types
   of tumor or cancer cell lines, including breast, lung, liver and
   stomach [[111]44, [112]45]. Overexpression of BUB1 and other family
   members has been found to be associated with tumor cell proliferation
   [[113]46]. Transgenic mice overexpressing BUB1 developed various
   spontaneous tumors, and showed accelerated myc-induced lymphomagenesis
   [[114]47]. High expression of BUB1 was observed in a variety of human
   malignancies including gastric cancer [[115]46, [116]48], colorectal
   cancer [[117]49], and lymphomas [[118]50]. We and others found high
   expression of MAD2L1 and BUB1 in breast cancer and their associations
   with unfavorable prognosis [[119]2]. Although mounting evidence
   suggests that high MAD2L1 or BUB1 are associated with tumor
   progression, study findings have been inconsistent. Reduced BUB1
   expression was observed in a subset of pancreatic cancer cells
   [[120]51]. This inconsistency may arise from different expression in
   these checkpoint components between actively proliferating cells and
   quiescent or differentiated cells. Increased BUB1 expression accords
   with higher mitotic index in tumors compared to neighboring tissues
   [[121]47]. Alternatively, variation in function may reflect
   compensation for other defects in the mitotic checkpoint.

   In our in vitro experiments, we also observed that down-regulation of
   MAD2L1 or BUB1 expression resulted in reduced cell migration and
   invasion. The underlying mechanism for these effects is unknown. One
   study showed that MAD2L1 was a direct transcriptional target of E2F,
   and their interaction could cause retinoblastoma pathway dysregulation
   [[122]52], which may lead to the suppression of migration and invasion.
   Another recent study found that knockdown of BUB1 could significantly
   inhibit the TGF-β-mediated induction of cell migration and invasion
   [[123]53].

   In summary, our study confirmed the prognostic value of two key mitotic
   checkpoint genes MAD2L1 and BUB1, which have been included in multiple
   gene expression signatures for breast cancer prognosis. We also found
   that these genes are biologically relevant to breast cancer
   progression, as suppression of their expression was associated with
   reduced tumor cell growth, migration and invasion. Together, our
   investigation suggests that these genes may serve as tumor markers for
   breast cancer prognosis as well as potential therapeutic targets to
   suppress tumor growth.

Supporting Information

   S1 Table. Summary of published gene signature included in the current
   study.

   (DOCX)
   [124]Click here for additional data file.^ (44.7KB, docx)
   S2 Table. List of genes appeared three or more gene signatures.

   (DOCX)
   [125]Click here for additional data file.^ (282.5KB, docx)

Data Availability

   The authors confirm that all data underlying the findings are
   available. GSE37751 dataset is available at the website
   [126]http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE37751.
   GSE29044 dataset is available at the website
   [127]http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE29044. For
   data from the Turin study, the nature of ethical restrictions is to
   protect patient data privacy and to ensure proper use of patient data
   for breast cancer research. Data will be available upon request to all
   interested researchers. Please contact the corresponding author at
   hyu@cc.hawaii.edu.

Funding Statement

   The authors received no specific funding for this work.

References