although the overall incidence of breast cancer is rising worldwide , the mortality rate has been decreasing in the united states .
the improved survival rate is likely to be a result of the success in early detection and better treatment in patients with positive estrogen receptors ( er ) , progesterone receptors ( pr ) , or human epidermal growth factor receptor 2 ( her2/neu ) breast cancers .
triple negative breast cancers ( tnbc ) by default have been grouped together because of the lack of er , pr , and her2/neu markers [ 4 , 5 ] .
compared to the other subtypes of breast cancer , these tumors are frequently more aggressive , manifested by a higher distant relapse rate with more frequent visceral as well as central nervous system metastases and higher mortality rate despite chemotherapy [ 68 ] .
the heterogeneous biology and histopathology of tnbc underlie the unpredictable responses to chemotherapy and diverse clinical outcomes seen in these patients .
the majority of tnbc with relapse is multidrug resistant and ultimately becomes refractory to all therapies [ 9 , 10 ] . to improve treatment , it is important to develop novel therapeutic strategies to predict and overcome drug resistance . in the last two decades ,
proteomics has emerged as a powerful tool in biomarker discovery and mechanism understanding . using these tools
proteomics has been used as a tool to identify new disease related biomarkers in tnbc [ 11 , 12 ] .
protein phosphorylation , one of the most ubiquitous posttranslational modifications ( ptms ) , is a key event in regulating many vital functions in cells including proliferation , survival , apoptosis , and signal transduction [ 1315 ] .
protein phosphorylation , involved in signal transduction of the cells , requires a coherent activation of protein kinases and phosphatases , which leads to the defined functions .
the basal level of the phosphor - proteins may also represent the characters of the cells .
reported that the stable tyrosine phosphorylation of the il-10 receptor may increase timp-1 levels to block tumor cell invasion in modified boyden chamber invasion assays .
brner et al .   reported that the stable phosphorylation of the inhibitory tyr-505 of the leukocyte - specific protein tyrosine kinase ( lck ) may arrest lck in its inhibited form . in recent years
, the advances in phosphoproteomics research have allowed discovery of many important functions operating in cancer progression .
performed quantitative phosphoproteome and transcriptome analysis on ligand - stimulated mcf-7 breast cancer cells to study the mechanism of tamoxifen resistance .
they found that gsk3 and ap-1 transcription factors might be involved in the tamoxifen resistance in mcf-7 cells .
used a phosphoproteomic approach to study lapatinib - resistance of her2-overexpressing human breast cancer cell lines and found that the increased src kinase activity was a mechanism of lapatinib resistance .
oliveras - ferraros et al . also reported a study on tnbc cell lines using low throughput phosphoproteomic approaches .
however , there has been no study focusing on dissecting tnbc drug resistance using large - scale phosphoproteomic tools . in this study , we used high throughput technologies to study changes in phosphorylated proteins to uncover important pathways involved in tnbc drug resistance .
for the purpose of this study , tnbc cell lines responding to multiple chemotherapeutic drugs were studied and were compared .
twelve established tnbc cell lines were tested against four chemodrugs and the half maximal inhibitory concentrations ( ic50s ) were calculated .
the phosphorylated peptides of four resistant and two sensitive cell lines were analyzed using lc - ms / ms to discover important pathways related to chemodrug resistance of tnbc .
our study may lead to identification of useful prognostic biomarkers and therapeutic pathways for tnbc treatment .
triple negative breast cancer cell lines mda - mb-231 , mda - mb-468 , mda - mb-436 , hcc1187 , hcc1806 , and hcc1937 ( all of which stain negative for er , pr , and lack her2/neu amplification ) were obtained from american tissue type culture collection ( atcc , manassas , va , usa ) [ 22 , 23 ] .
cells were maintained in dulbecco 's minimal essential medium ( invitrogen , carlsbad , ca , usa ) or rpmi 1640 ( invitrogen ) with 10% heat - inactivated fetal bovine serum ( thermo fisher scientific , san jose , ca , usa ) , 100 units / ml penicillin , and 100  g / ml streptomycin , at 37c in 5% co2 .
cell lines were treated with docetaxel , doxorubicin , gemcitabine , and cisplatin in vitro to determine the half maximal inhibitory concentration ( ic50 ) of each drug .
the cells were treated with dmso or twenty predetermined doses of each drug for two days .
cell viabilities were determined by celltiter - glo luminescent cell viability assay ( promega , madison , wi , usa ) .
triplicated experiments were performed twice and the ic50s were calculated using graphpad prism 5 software .
cultured cells were lysed in lysing buffer ( 8  m urea , 4% chaps , 40  mm tris - base , 65  mm dtt , 1% sds ) and the supernatant was collected into 1.5  ml tubes .
protein concentration of the lysate was determined using the pierce bca protein assay ( thermo fisher scientific ) .
20  l of 1  m dtt ( thermo fisher scientific ) was added to samples containing 1  mg of proteins and incubated at 56c for 1 hour and followed by an incubation at room temperature for 40  min in darkness with 80  l of 1  m iaa added into the buffer .
each sample was treated with 0.11 volumes of ice - cold 100% trichloroacetic acid ( tca ) ( sigma aldrich , st .
louis , mo , usa ) on ice for 10  min and 500  l of ice - cold 10% tca on ice for 20  min and then was spun down at 20,000  g for 30  min .
the pellet was washed with 500  l of acetone and was centrifuged again at 20,000  g for 10  min .
500  l of 100  mm ammonium bicarbonate ( sigma aldrich ) and 20  g of trypsin ( promega , san luis obispo , ca , usa ) were added to the sample in each tube at 37c for 2 hours .
an additional 20  g of trypsin was added to the sample and subsequently incubated for 16 hours .
the peptides were then filtered through 10  kda filter columns ( emd millipore , billerica , ma , usa ) and dried via vacuum evaporator .
400  l of loading buffer ( 80% acetonitrile ( acn ) and 2% trifluoroacetic acid ( tfa ) ) was added to 1  mg of peptides .
the mixture was incubated with 4  mg of tio2 beads ( gl sciences , torrance , ca , usa ) for 1 hour .
the samples were centrifuged at 3,000  g for 5  min and the supernatant was discarded .
tio2 beads were collected and washed with 1  ml wash buffer i ( 30% acn , 2% tfa ) followed by 1  ml of wash buffer ii ( 80% acn , 0.1% tfa ) , each for 20  min at 4c with rotation and centrifuged at 3,000  g for 5  min .
the phosphopeptides were eluted first with 400  l elution buffer i ( 400  mm nh4oh , 50% acn , ph 11 ) and was followed by 400  l elution buffer ii ( 500  mm nh4oh , 60% acn , ph 11 ) .
nest group microspin strong cation exchange solid phase extraction tubes ( the nest group inc . , southborough , ma , usa ) were used as a separation technique before mass spectrometry to reduce the complexity of samples and enhance the identification rate .
all peptide fractions were desalted before analysis using c18 tips made from the empore c18 90  mm disk ( 3  m corporate , st .
nanoliquid chromatography and tandem mass spectrometry ( nlc - ms / ms ) with collision induced dissociation ( cid ) was performed on a ltq - orbitrap ( thermo fisher scientific ) integrated with an eksigent nano - lc ( eksigent technologies , monmouth junction , nj , usa ) .
the flow rate for reverse - phase chromatography was 500  nl / min for the loading and analytical separation ( buffer a : 0.1% formic acid , 3% acn ; buffer b : 0.1% formic acid , 100% acn ) .
the orbitrap was operated in data - dependent mode with a full precursor scan at high - resolution ( 60000 at m / z 400 ) and ten ms / ms experiments at low resolution on the linear trap while the full scan was completed . for cid
mass spectra were searched against the uniprot human database using proteome discoverer software ( version 1.4 , thermo fisher sceintific ) , utilizing the sequest ( thermo fisher scientific ) , mascot ( matrix science , london , uk ) , and x !  tandem ( http://www.thegpm.org/tandem/ ) algorithms , while running a target decoy search strategy to increase protein identity confidence .
mascot and sequest were searched with a fragment ion mass tolerance of 0.80  da and a parent ion tolerance of 10.0  ppm and tolerated up to two missed trypsin cleavages .
glu or gln to pyro - glu of the n - terminus , oxidation of methionine , and phosphorylation of serine , threonine , and tyrosine were specified as variable modifications .
the proteome discoverer search results were loaded to scaffold ( version 4.1.1 , proteome software inc . , portland , or , usa ) to quantify and validate the ms / ms peptide and protein identifications .
identifications were accepted if they had a greater than 95% peptide probability and contained at least one identifiable phosphopeptide .
phosphosite localization probabilities were calculated using the ascore probability based scoring technique   and only sites that met the stringent minimum of 99% were accepted .
the spectral count data of the phosphopeptides were acquired through scaffold ptm and were compared between grouped cell lines : ( 1 ) all four resistant cell lines were compared with the two sensitive cell lines hcc1806 and mda - mb-468 ; ( 2 ) resistant cell lines mda231 and mda - mb-436 were compared to the two sensitive cell lines ; ( 3 ) resistant cell lines hcc1187 and hcc1937 were compared to the two sensitive cell lines .
a t - test of each peptide between the groups of the three comparisons was performed and the peptides with p values < 0.05 and a fold change of at least 2.0 ( 2.0 or 0.5 ) were considered differential peptides . from the comparison ( 1 ) ,
all changed phosphopeptides were further analyzed using online database string which is a database of known and predicted protein interactions including direct ( physical ) and indirect ( functional ) associations .
hierarchical clustering analysis was performed using euclidean distance formulation and complete linkage criteria for linkage of normalized phosphopeptide spectrum counts .
based on the comparison ( 1 ) , only the peptide spectrum counts with p values less than 0.05 were imported to permutmatrix software .
protein phosphorylation is an important posttranslational modification that governs many of the signaling changes in cancer cells .
the current study was performed to screen the signaling pathway changes , through the comparison of phosphorylated proteins in tnbc cell lines with extreme responses to the four chemotherapy drugs .
the half maximal inhibitory concentrations ( ic50s ) of twelve tnbc cell lines against four chemotherapy drugs were determined and four resistant and two sensitive cell lines were selected for further analysis .
cell lines were ranked according to their ic50s against each drug ( table 1 ) .
no response is defined as the inability to reach a 50% of inhibition when the highest dose of chemotherapeutic drug was administered .
coefficient of determination r values of the drug sensitivity curves ( data not shown ) less than 0.7 were also considered as no response .
hcc1187 , mda - mb-436 , mda - mb-231 , and hcc1937 had the highest ic50s for at least three of the four drugs and were thus considered chemotherapy - resistant .
hcc1806 and mda - mb-468 were ranked lowest on the ic50 scale for at least three of the four drugs and were considered chemotherapy - sensitive .
these six cell lines were selected for phosphorylated protein analysis . to increase the accuracy of identification ,
the spectra were cross - validated using three searching algorithms ( sequest , mascot , and x ! 
tandem ) against the uniprot - human database with the application of a decoy database .
a total of 1,645 phosphoprotein groups were identified by scaffold software across all six tnbc cell lines , using peptide probability thresholds of 95% with a minimum of one unique peptide ( figure 1(a ) ) . the peptide false discovery rates ( fdr )
accordingly , a total of 9,585 unique phosphopeptides with 10,091 unique phosphosites were identified ( see supplementary table  1 in supplementary material available online at http://dx.doi.org/10.1155/2014/390781 ) .
instances where the same peptide sequence had different phosphosites were counted as one peptide and ultimately 3,062 peptide sequences were identified ( figure 1(b ) ) .
the overall class profiles of the phosphoproteins specifically for resistant and sensitive cell lines were also expressed according to their gene ontology ( go ) annotation . when compared to each other ,
more phosphoproteins involved in cellular adhesion processes were found in resistant cell lines while in sensitive cell lines more phosphoproteins were associated with multiorganism processes , cell killing , pigmentation , and rhythmic processes ( figures 1(c ) and 1(d ) ) .
when the cellular compartments of these proteins were studied , the resistant cell lines showed more phosphoproteins in extracellular regions , mitochondrion , and plasma membrane whereas in sensitive cell lines more phosphoproteins were found in golgi apparatus and cytoskeleton ( figures 1(e ) and 1(f ) ) .  
figure 2 shows the representative ms / ms spectra for three phosphopeptides marked with phosphosites .
these phosphopeptides differed dramatically between drug resistant and sensitive cell lines and will be discussed below .
the high quality spectra maps were very helpful in identifying peptides as well as phosphosites on them . in the current study ,
scaffold ptm was used to further confirm the phosphosites identified and to construct a final quantification report .
scaffold ptm uses the ascore algorithm to verify the presence of the phosphorylation sites . with the condition of a 99% ascore certainty and 99% minimum localization probability threshold , a total of 1,340 phosphoproteins groups ,
2,760 unique phosphopeptides , and 4,549 unique phosphosites were identified across all 6 cell lines ( supplementary table  2 ) .
the mass spectra quantification data of these phosphopeptides were exported and further analyzed to determine the variations between chemotherapy - resistant and chemotherapy - sensitive cell lines ( supplementary table  2 ) .
the spectrum counts of phosphorylated peptides were evaluated by computing the p value and fold change between the two groups of cell lines . only phosphopeptides with a p value less than 0.05 and a fold change above 2 or less than 0.5 were considered . from three sets of comparisons ( seen in section 2 ) , three sets of numbers
were obtained ( shown in supplementary table  3 , sheets 1 , 2 , and 3 ) .
all significantly changed phosphopeptides were further analyzed using the online string database for predicted protein interactions ( see below ) .
four drug resistant cell lines and two drug sensitive cell lines selected by ic50 ranking were subjected to phosphoproteome analysis in the current study . to confirm that these cell lines can be separated by the differentially expressed phosphopeptides into two groups : chemotherapy - resistant and chemotherapy - sensitive tnbc , the clustering analysis was performed .  
figure 3 shows that the four drug - resistant cell lines shared much more similarity with each other than the two sensitive cell lines and they could be clearly segregated from the two sensitive cell lines .
this result further supported the ic50 data and suggested that the phosphopeptide identification and quantification methods are valuable in characterizing drug sensitivity of tnbc .
the changed phosphopeptides described above were then analyzed with string database . all the changed phosphopeptides from supplementary table  3 were analyzed .
the corresponding genes of these phosphopeptides were loaded to string to construct a network showing the associations between them .
the genes with most connections in the network were shown in figure 4 and table 2 .
as shown in figure 4 , the changed phosphoproteins have strong associations and intricate interactions with one another .
the pathways associated with the most prominent changes in drug - resistant tnbc are schematically summarized in figure 5 .
over the last few decades there has been a steady decrease in breast cancer mortality rate largely due to the improvements in the treatment of breast cancer . despite the significant advancement made in breast cancer therapy in recent years
, much of this progress is limited to hormone receptor positive and her2/neu positive breast cancers .
specific targeted agents are still lacking for tnbc tumors leaving cytotoxic chemotherapy as the only therapeutic choice . though cytotoxic chemotherapy is effective for many of these patients , the absence of a therapeutically targetable molecule or pathway limits the progress in treating these patients , which is manifested by rapid relapse and death when the tumors are resistant to the conventional chemotherapy .
the identification of novel biomarkers indicative of drug sensitivity is imperative for the effective treatment of this exceptionally aggressive type of breast cancer . in our previous study
we utilized a hydrophobic fractionation protocol in an effort to detect novel membrane proteins in triple negative breast cancer .
although the identification of membrane biomarker is valuable , the heterogeneity of tnbc tumors commands the identification of a variety of biomarkers and signaling pathways in order to arrive at the best treatment strategy .
it is important to identify aberrations in tnbc subtypes that cause these tumors to be resistant and unresponsive to chemotherapeutic treatments .
protein phosphorylation plays a crucial role in cellular signal transduction pathways . when activated , protein kinase binds and phosphorylates a specific substrate to mediate preprogrammed protein function [ 29 , 30 ] . to find potential biomarkers that could predict the patient 's response to chemotherapy and potential targets to improve tnbc treatment , we profiled phosphorylated peptides in tnbc cell lines with different sensitivities to a variety of chemotherapy drugs .
label - free quantification was favored in this study since it has the largest dynamic range and highest proteome coverage - a prerequisite for the objective of this study as protein phosphorylation is a transient process and can be found with varying concentrations [ 31 , 32 ] .
fractionation via scx was also utilized in this study to decrease the sample complexity and thus improve the identification .
software as well as several computer - based algorithms was also used to correct any inconsistencies and to increase reliability , thus allowing for more accurate results .
as shown in figure 3 , the phosphopeptide clustering data perfectly matched the drug sensitivity data ( table 1 ) .
these results gave additional proof that the quantification and identification system used was reliable and potentially had predictive value . because different phosphosites in a protein may trigger either protein activation or inactivation , we used phosphosite instead of phosphoprotein as unit for quantification .
the functions of proteins will be discussed according to the changes of phosphosites . among the dramatically altered phosphoproteins identified in the current study , many
our results further support the roles of these proteins in tnbc drug resistance and offer new insights for future studies .
some new phosphorylated sites found in this study together with several of the previously reported protein phosphosites were connected for the first time to tnbc drug resistance .
transcription factor ap-1 ( ap-1 ) is a multiprotein transcription factor and a member of the jun and fos protoontogenetic family .
previous studies have linked the activated ap-1 and its family members to increased tumorigenesis , metastasis , and invasion [ 33 , 34 ] as well as drug resistance [ 35 , 36 ] .
overexpression of phosphorylation at ser73 of c - jun was reported to be responsible for the development of multidrug resistance in colorectal cancer cells . in the current study
phosphorylated ser73 of c - jun was only found in the resistant cell lines , suggesting ap-1 is important in tnbc drug resistance .
the overexpression and activation of promyelocytic leukemia protein ( pml ) is known to induce the transcriptional activation of ap-1 [ 38 , 39 ] . in this study
, we also found an upregulation of ser583 on pml in drug resistant cell lines , a new phosphosite which has not been previously described .
our data suggests that this phosphosite provokes the activation of pml , which may lead to tnbc drug resistance through the activation of ap-1 .
heat shock factor 1 ( hsf-1 ) is a master regulator for the transcription and heat shock proteins ( hsps ) and was reported to induce a multidrug resistance phenotype through constitutive activation of the multidrug resistance gene 1 ( mdr-1 ) . in the current study phosphorylation of ser303 on hsf-1 was identified to be upregulated in the resistant cell lines .
proposed that phosphorylation of ser303 induced a slow repression of hsf-1 allowing for the accumulation of the hsps that are crucial for cell growth and recovery .
the above evidence suggested that ser303 phosphorylation of hsf-1 might play an important role in tnbc drug resistance .
in addition , both hsf-1 and ap-1 were capable of activating vimentin gene and could be responsible for its overexpression [ 4244 ] .
vimentin is a well - recognized biomarker in epithelial to mesenchymal transition ( emt ) and has been associated with metastasis , poorer prognosis , and cell motility in various types of cancer . in the current study , ser73 and ser56 on vimentin
were found to be phosphorylated in the resistant tnbc cell lines but were absent in the sensitive cell lines .
interestingly , cyclin - dependent kinase 5 ( cdk5 ) was reported to mediate phosphorylation at ser56 of vimentin .
ser23 of lamin b1 ( lmnb1 ) was also identified as an upregulated phosphosite in the resistant cell lines in this study .
taken together , cdk5 might be activated in drug resistant tnbc cell lines and might phosphorylate lmnb1 and vimentin , leading to emt and drug resistance in tnbc .
it has been reported that epidermal growth factor receptor ( egfr ) plays a significant role in hepatocyte growth factor receptor ( hgf / c - met ) mediated biological activities .
activation of hgf is indicative of aggressive tumor pathology , including enhanced proliferation and invasion .
egfr might also promote the multiple drug resistance ( mdr ) phenotypes in breast cancer cells via accelerating the g1/s transition .
furthermore , bagowski et al . reported that egfr phosphorylation at thr654 and thr669 by prkd1 inhibits egf - induced mapk8/jnk1 activation .
their studies showed that activation of egfr and hgf might contribute to drug resistance of breast cancer through the downregulation of phosphosites such as thr654 and thr669 of egfr .
in the current study , the phosphosites thr-693 , tyr-1197 , and tyr-1110 on egfr and thr977 on hgf were found to be downregulated in resistant cell lines , suggesting a release of negative regulation of egfr and hgf functions in these cells .
downregulation of these phosphosites might increase egfr and hgf activity and led to drug resistance of tnbc cell lines .
it is noticeable that many of the changed phosphoproteins found in the current study were involved in mrna processing including heterogeneous nuclear ribonucleoprotein a2/b1 , serine / arginine repetitive matrix 1 and matrix 2 , eukaryotic translation initiation factor 4 gamma-1 , and several others . as reviewed by eblen ,
alternative splicing is a normal cellular process that can be manipulated by a cancer cell to enhance survival in response to chemotherapeutic treatment .
we also found the changes of phosphorylation status of some microtubule related proteins and dna binding proteins in the current study ( figure 4 and table 2 ) .
the current study has shown that proteomic analysis is a powerful tool for profiling of the phosphorylation patterns and may help better understand drug resistant tnbc cells .
our data suggested that pml , ap-1 , and hsf-1 were preferentially activated in resistant tnbc cells and might promote downstream signals including vimentin activation .
in addition , our study also suggested that cdk5 might also promote vimentin and lmnb1 activation leading to an increased emt in the resistant tnbc cells .
we are also reporting on the potential roles of egfr and hgf in the promotion of a multiple drug resistance ( mdr ) phenotype in tnbc .
we have identified several signaling pathways that may work together to mediate the drug resistance in tnbc ( figure 5 ) .
 because of the absence of a clear therapeutic target for triple negative breast cancer ( tnbc ) , conventional chemotherapy is the only available systemic treatment option for these patients . despite chemotherapy treatment , tnbc patients still have worse prognosis when compared with other breast cancer patients . the study is to investigate unique phosphorylated proteins expressed in chemoresistant tnbc cell lines . in the current study 
 , twelve tnbc cell lines were subjected to drug sensitivity assays against chemotherapy drugs docetaxel , doxorubicin , gemcitabine , and cisplatin . based on their half maximal inhibitory concentrations , 
 four resistant and two sensitive cell lines were selected for further analysis . 
 the phosphopeptides from these cells were enriched with tio2 beads and fractionated using strong cation exchange . 
 1,645 phosphoprotein groups and 9,585 unique phosphopeptides were identified by a high throughput lc - ms / ms system ltq - orbitrap . 
 the phosphopeptides were further filtered with ascore system and 1,340 phosphoprotein groups , 2,760 unique phosphopeptides , and 4,549 unique phosphosites were identified . our study suggested that differentially phosphorylated cdk5 , pml , ap-1 , and hsf-1 might work together to promote vimentin induced epithelial to mesenchymal transition ( emt ) in the drug resistant cells . 
 egfr and hgf were also shown to be involved in this process . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion