BACKGROUND
oestrogen-receptor  negative breast cancer  accounts for approximately 20% of all newly diagnosed breast malignancies  <cit> .
clinically, however, this group of bcs contains different subtypes and can be subdivided into either her2-positive or triple-negative bcs, defined by very low or absent immunohistochemical expression of er and progesterone receptor , and low expression and lack of amplification of her <dig>  <cit> .
triple-negative bcs account for 10-15% of all breast tumours and are mostly of high grade, have a high incidence of tp <dig> mutations, and show proliferative characteristics with a higher propensity to spread to visceral organs  <cit> .
sharing many of these phenotypic features with triple-negative bcs are breast tumours of the ‘intrinsic’ basal-like subtype.
these tumours generally lack er and her <dig> expression and are molecularly characterised by the expression of genes associated with both basal epithelium and myoepithelium of the normal mammary gland   <cit> .
approximately, 80% of triple-negative bcs show features of basal-like bcs  <cit> .
while most triple-negative bcs show aggressive clinical behaviour and have very limited targeted therapies, they also encompass subgroups of cancers sensitive to chemotherapy and having a good prognosis  <cit> .
hence, continuous efforts to characterise this bc population have already identified several subgroups.
one of the proposed groups comprise “claudin-low” tumours, which are characterised by gene expression profiles similar to those found in the so-called breast ‘cancer stem cell’ populations  <cit> , while other subgroups were classified as having higher expression of the interferon-related or apocrine genes  <cit> .
bc cell lines are essential tools in bc research and have been widely used to elucidate bc biology and new therapies  <cit> .
since cell lines are easily propagated and genetically manipulated, extensive information about their transcriptome, genome and to a lesser extent epigenome has been produced  <cit> .
several studies have compared and integrated gene expression profiles and genomic alterations between primary breast tumours and bc cell lines, demonstrating that the heterogeneity found in primary bcs is to a certain extent recapitulated in the panel of commonly used bc cell lines  <cit> .
given the increasing knowledge of the diversity and complexity among bc subtypes it has also become evident that no individual cell line will recapitulate all aspects of the disease.
here we interrogated genome-wide transcriptional profiles with genomic and epigenetic profiling in a collection of  <dig> bc cell lines enriched for those of triple-negative phenotype.
we have focused on gene signatures, underlying dna copy number aberrations  and epigenetic events specifically associated with triple-negative bcs.
by cataloguing these perturbations on a gene-centric basis we have extended the characterisation of these bc cell lines and offer valuable insights on their suitability in modelling certain features of this heterogeneous disease.
RESULTS
bc cell lines segregate into three groups based on their transcriptional profiles
to investigate the molecular heterogeneity of triple-negative bc cell lines and their representativeness of triple-negative breast cancers, we used illumina humanwg-6v <dig>  to survey the phenotypic and genotypic characteristics of seven er-negative mesenchymal bc cell lines  and compared them with  <dig> er-negative epithelial-like bc cell lines .
five er-positive bc cell lines  were also included in our dataset, to evaluate er-responsive transcriptional signatures in er-negative bc cell lines.
in addition, two er-negative/ her2-positive epithelial bc cell lines were included , and were employed as comparators with other triple-negative bc cell lines.
molecular pathological features of all bc cell lines are provided in additional file  <dig> table s <dig>  unsupervised hierarchical clustering of  <dig>  highly variable ensembl genes separated er-negative bc cell lines into three groups .
one group, designated “cluster 1”  included three er-negative/her2-positive bc cell lines, namely skbr <dig>  sum <dig> and sum <dig>  which clustered with er-positive/her2-negative  and er-positive/her2-positive  cell lines.
“cluster 2” was uniformly composed of er-negative/her2-negative bc cell lines  and was in complete concordance with the basal “b” bc cell line subtype described in previous bc cell line studies  <cit> .
“cluster 3” consisted of cell lines  either having a triple-negative phenotype or showing amplification and higher abundance of her <dig>  and egfr gene amplification .
investigating the expression levels of known triple-negative bc-related genes demonstrated subtle differences within these three groups.
while “cluster 3” cell lines expressed genes commonly found in the intrinsic gene list preferentially expressed in basal-like primary bc   <cit> , “cluster 2” was characterised by a higher abundance of cav <dig>  a marker for the basal-like phenotype of sporadic and hereditary breast cancers  <cit> , and the lymphangiogenic factor vegfc, a potential therapeutic target for triple-negative bcs  <cit>  .
other genes previously associated with triple-negative bcs such as c-met, cd <dig> and cav <dig> <cit>  exhibited higher expression in both groups in comparison to “cluster 1” cell lines .
representation of er-negative breast tumour-related gene signatures in bc cell lines
given that the combined expression pattern of certain genes can uniquely characterise different bc subtypes or can be used as surrogate markers for pathway activation, we selected a compendium of gene signatures representing various features of triple-negative bcs.
firstly, we investigated the representation of eleven gene signatures based on a “relative similarity score” and ranked the bc cell lines accordingly .
overall “cluster 3” cell lines  showed a good representation of gene signatures identified for “apocrine”, g <dig> tn.tumour” and high-grade breast carcinomas, while “cluster 2” cell lines exhibited high similarities with the “stroma”, “mammosphere” and “cd <dig> cd44” expression patterns .
the resemblance to the remaining four gene signatures showed a less striking concordance with the gene expression defined clusters, and identified sum <dig>  hcc <dig>  sum <dig> and hcc <dig> cell lines as the best representatives for the “met”, “egfr”, “igf1” and “interferon” gene signatures, respectively.
secondly, we examined the  <dig> different er-negative bc subgroups associated with a prognostic outcome  <cit>  in the bc cell line transcriptomes based on centroid correlation classification.
as shown in figure 2b, “cluster 3” cell lines had activation of the “cell cycle and cell proliferation pathway/immune response genes” and the “cell cycle and cell proliferation pathway” groups , both of which showed association to basal-like bcs in their study  <cit> .
in contrast, “cluster 1” cell lines displayed highest correlation to er-negative tumours of the steroid response group, supporting the outcome of our hierarchical clustering in which these cell lines group with the er-positive cell lines .
lastly, to explore the representation of the molecular basal-like bc subtype within this bc cell line set, we performed nearest centroid classification using the class centroids from parker et al.
<cit> , sorlie et al.
<cit> , hu et al.
<cit> , prat et al.
<cit>  and guedj et al.,  <cit>  .
as expected given the manner in which the “claudin.low” gene signature was originally established  <cit> , “cluster 2” cell lines  are representatives of the described “claudin.low” subtype.
the recently published cit classification  <cit>  assigned all “cluster 2” and “cluster 3” cell lines to the basall group  with the exception of hs578t which was assigned to the “luminala” subtype.
interestingly, in “cluster 1”, er-negative/her2-positive cell lines skbr <dig>  sum <dig> and sum <dig> were labelled as mapo  groups, in agreement with er-negative/her2-positive breast carcinomas in this molecular subtype  <cit> .
pam <dig> classification assigned all “cluster  <dig> and 3” bc cell lines except hs578t and hcc <dig> to the basal-like bc subtype.
assignments of cell lines into the basal-like subtype with class centroids obtained from sorlie <dig> and hu <dig> were in good agreement with those obtained with pam <dig>  however only  <dig> out  <dig> cell lines were consistently classified as of luminal a, luminal b or her <dig> by all methods.
to determine the agreement of these three centroid classifications, we used the free-marginal kappa statistics of brennan and prediger  <cit>  and saw substantial agreement in the classification of the basal-like , luminal a  and her <dig> subtypes , in agreement with the results of previous studies  <cit> .
copy number aberrations and associated gene expression changes in bc cell lines represent those observed in triple-negative bcs
to identify bc cell lines harbouring copy number aberrations specific for triple-negative bc, we performed acgh using a 32k tiling path array platform and surveyed their genomic changes .
by grouping the bc cell lines based on the three expression clusters, “cluster 2” cell lines displayed significantly less high-level amplifications and deletions compared with the other two clusters .
next, we retrieved cnas identified in our previous study on  <dig> triple-negative bcs  <cit> , analysed on the same genomic platform.
overall the frequency of gains and losses seen in triple-negative bcs was more similar to “cluster 3” than to “cluster 2” and “cluster 1” cell lines .
recurrent amplification seen in primary triple-negative bcs were recapitulated in at least one bc cell line .
the most highly recurrent triple-negative bc-specific amplicons in bc cell lines were on 5p <dig> -p <dig>  , followed by 9p <dig> - <dig>  and 7q <dig>  found in  <dig> “cluster 3” cell lines and  <dig> “cluster 3” cell lines, respectively .
given that these regions are also characterised by common polymorphisms, genes such as jak <dig>   <cit> , nuns <dig>   <cit>  or limk <dig>   <cit>  previously associated with breast cancers, and gained preferentially in basal-like breast cancers  <cit>  might validate that these regions contain genes providing a selective advantage for triple-negative bcs.
a comprehensive integration of genes lost or gained in the  <dig>  breast cancer study is provided in additional file  <dig> table s <dig>  amplifications on 3q24-q <dig> , 3q <dig> -q <dig> , 5p <dig> -p <dig> , 7p <dig>  and 9p <dig>  were observed only in ”cluster 3” cell lines, and the 13q <dig> -q <dig>  amplicon only in “cluster 2” cell lines, illustrating a good representation of triple-negative bc-specific cnas in er-negative cell lines, with some of them having a higher prevalence in one than the other expression defined group.
we and others have previously demonstrated that the expression levels of certain genes located in triple-negative bcs specific cnas is copy number dependent  <cit> .
to investigate if these dependencies are also recapitulated in bc cell lines, we integrated expression data with cbs-smoothed acgh profiles of each bc cell lines.
using pearson’s correlation ,  <dig>  genes showed significantly correlation between their expression and dna copy number levels .
this set encompassed  <dig> / <dig>  triple-negative bc copy-number dependent genes as determined by turner  <cit> , and included genes such as transcriptional regulators , kinases , phosphatases  and transmembrane receptors , as well as biomarkers for diagnosis , prognosis , disease progression  and known drug targets  .
triple-negative bc–specific amplicons, recurrently amplified in our bc cell lines  and harbouring genes with dna copy number-dependent expression levels are shown in figure  <dig>  among those were genes, such as jak <dig>  nsun <dig> and nfib previously shown to have pathogenic roles, but also novel potential drivers e.g.
ppapdc or ranbp <dig>  which could be selectively required for the survival of cells harbouring those amplification .
epigenetic influence on sub-type specific genes in er-negative bc cell lines
the functional validity of the methylation pattern found on cpg islands in cultured cancer cell lines has been the matter of controversy  <cit> .
to study epigenetic patterns in bc cell lines, we produced genome-wide methylation profiles on illumina goldengate bead arrays.
unsupervised hierarchical clustering using  <dig>  cpg probes, corresponding to  <dig> genes, revealed a different grouping  as it was observed when their expression profiles were clustered .
to validate that the bc cell line specific methylation pattern can be also found in triple-negative bc, we retrieved methylation data of  <dig> fresh-frozen bcs performed on the same microarray  <cit> .
due to the lack of her <dig> status information in their study,  <dig> basal-like bcs were used as surrogates for triple-negative tumours and  <dig> cpg islands with variable methylation levels were observed.
of those  <dig>   <dig> cpg probes showed also changes in their methylation status in our bc cell lines .
investigating the methylation state of these cpg islands in each bc cell line individually, demonstrated an overall good representation of ≥70% of hypo- and hyper- basal-like bc-specific methylation events specifically in er-negative bc cell lines .
given that bc subtype specific expression has been suggested to be under epigenetic influence  <cit> , we surveyed the methylation effect on gene expression in bc cell lines.
integration of methylation and gene expression data resulted in  <dig>  cpg gene pairs  and identified an inverse correlation between methylation and gene expression levels for  <dig> pairs .
performing bootstrap analysis by randomly sampling the bc cell lines, we showed that the number of significant association between gene expression and methylation was more than  <dig> fold higher than expected.
using these  <dig> cpg gene pairs in a multiclass sam analysis revealed  <dig> with specific methylation patterns over the three expression clusters, particularly distinguishing “cluster 2” cell lines from the others .
as described previously, “cluster 2” cell lines exhibited among others, expression patterns similar to the “claudin.low” gene signature  <cit> .
fifteen genes of the “claudin.low” gene signature had cpg sites with varying methylation pattern over these bc cell lines which was significantly higher than expected by chance , whereby genes downregulated in “claudin.low” cancers according to “claudin.low” signature were methylated and vice-versa, such as “claudin.low” signature genes such as prss <dig>  cldn <dig> and vamp <dig> were downregulated in “cluster 2” and their cpg islands were methylated, while genes like sparc or ddr <dig> had unmethylated cpg islands and showed higher abundance in these bc cell lines.
by comparing those with epigenetic-regulated genes in breast cancers  <cit> , we identified  <dig> genes with the same concordant pattern .
taken together, our analyses demonstrate that bc cell lines retain methylation–dependent gene expression patterns observed in basal-like bcs, and strengthen an epigenetic influence on some bc phenotypes that are retained in their equivalent model systems.
discussion
triple-negative bcs represent a heterogeneous group with diverse deregulation of biological pathways.
here, we extended the molecular characterisation of er-negative bc cell lines based on their genetic, epigenetic and transcriptional profiles, and correlated these with a comprehensive compendium of gene signatures reflecting different features of er-negative bcs  <cit> .
initial cluster analysis of bc cell lines’ expression profiles resulted in three groups, two clusters encompassing purely er-negative bc cell lines , while one consisted of three er-negative and all er-positive bc cell lines.
the first two cell line clusters were in good agreement with recent bc cell line studies  <cit> .
while “cluster 2” encompassed cell lines that were all represented in the basal “b” cluster of neve et al.
<cit>  and were assigned to the triple-negative mesenchymal phenotype by lehmann et al.
<cit> , most of the “cluster 3” cell lines were part of neve’s basal “a” cluster  <cit>  and part of the basal-like subtype according to lehmann et al.,  <cit> .
our “cluster 3” cell lines exhibited expression patterns found in transcriptional profiles of microdissected grade  <dig> triple-negative breast tumours  <cit>  as well as grade  <dig> versus grade  <dig> breast carcinomas  <cit> .
hcc <dig>  an er-negative/her2-negative cell line, was the top in vitro representative “cluster 3” cell line for the triple-negative phenotype of microdissected grade  <dig> triple-negative breast tumours  <cit> .
the transcriptional profile of hcc <dig> also seemed very suitable in modelling the interferon, igf <dig> and met signalling pathways.
bc cell lines with expression patterns most closely associated with the apocrine.basal subtype  <cit>  were not defined to one or the other cluster and hcc <dig>  an er-negative/her2-positive cell line of “cluster 3” displayed the highest representation.
these bcs were originally defined on the basis of their androgen receptor level and many of them harboured erbb <dig> amplifications  <cit> .
this is in agreement with our findings, whereby using a recently published bc classifier, named cit, three er-negative/her2-positive cell lines skbr <dig>  sum <dig> and sum <dig> were classified to the mapo  breast cancer subtype  <cit> .
in a study, mdamb <dig>  sum <dig>  cal <dig> and mfm <dig> showed expression patterns associated with androgen receptor signalling and were more sensitive to androgen receptor antagonist bicalutamide and an hsp <dig> inhibitor  <cit> .
while none of those cell lines were part of our study, bt <dig> and hcc <dig>  bc cell lines used in our study and good representatives of the apocrine.basal subtype showed high sensitivity to hsp <dig> inhibitors in lehmann’s work  <cit> .
the claudin-low subtype has been described as bc entity  <cit> , which is enriched for er-negative invasive ductal carcinomas, while displaying low levels of luminal differentiation markers and activation of pathways involved in epithelial-to-mesenchymal transition, stem cell-like features and the immune response  <cit> .
integration of gene expression with methylation data over bc cell lines revealed a group of cpg islands corresponding to genes within the claudin-low signature, showing an inverse correlation between their methylation and the genes expression in bc cell lines and bcs  <cit> .
our findings are in agreement with those from a recent report that led to the identification of a set of genes whose expression was epigenetically regulated and when used as a gene signature identified mesenchymal features in claudin-low breast tumours  <cit> .
furthermore, they postulated that a deviant methylation might reflect cell lineage commitment in agreement with our hypothesis of a contribution of an epigenetic regulation to the claudin-low subtype.
aberrant dna methylation events have initially been thought to accumulate in a random fashion within cells in pre-malignant tissues, however, lately it has also been shown that de novo methylation has a predictable pattern, creating plasticity followed by commitment to alternative cell lineages  <cit> .
holm and colleagues proposed that bc subtypes might be driven by different epigenetic events and could reflect their different cellular origins  <cit> .
nevertheless, an alternative hypothesis might also be that the methylation patterns are a result from mutations in genes controlling the epigenetic landscape in breast cancer  <cit> ; thus further investigation is warranted to determine whether these distinctive methylation patterns are results of genetic aberration in epigenetic regulator genes and/or contribute to delineation of the differentiation hierarchy of claudin-low and other bc subtypes.
we and others have recently shown that basal-like bcs are most likely derived from luminal progenitor cells  <cit> .
identifying in vitro models would enhance our understanding of these cell populations.
interestingly, our cluster and gene signature analysis revealed er-responsive features for skbr <dig>  sum <dig> and sum <dig>  three er-negative/her2-positive cell lines.
skbr <dig> cells are well known to have luminal bc characteristics  <cit> .
in contrast, the classification of sum <dig> and sum <dig> is controversial.
while some bc cell line studies assigned them to basal-like cell lines  <cit> , others supported our finding of sum <dig> within the er-positive cluster  <cit> .
sum <dig>  although not included in this study, was classified as of luminal phenotype in other studies  <cit> .
common to both is the expression of luminal cytokeratins  <dig>   <dig> and  <dig>  <cit>  as well as genes found in luminal progenitor cell population   <cit> , more consistent with a luminal classification.
although sum <dig> was found to highly express aldh <dig>  a marker for the so-called bc stem cells  <cit> , further investigations are necessary to ascertain whether sum <dig> and sum <dig> represent appropriate in vitro models for luminal intermediate progenitor populations.
high-level amplifications are less likely to represent random aberrations and often encompass genes driving the development or maintenance of tumour growth.
three-quarters of triple-negative bcs harbour at least one amplicon  <cit> , however, their recurrence rates are lower than those of high-level cnas found in er-positive/ her2-negative and her2-positive bc subtypes .
here, we demonstrated that triple-negative bc-specific amplicons are recapitulated in er-negative bc cell lines and that some of them are associated with higher frequencies either to ”cluster 2” or “cluster 3” expression clusters.
for example, the region on 5p <dig> -p <dig>  was found to be recurrently amplified in 5/ <dig> and 10/ <dig> triple-negative bcs  <cit> , was present in six ”cluster 3” but only in one ”cluster 2” cell lines.
notably, these genomic sites map to regions of common germline copy number polymorphism and the functional consequences of their increased dna levels require further validation.
nevertheless, several genes located within these amplified regions were found gained with a higher frequency in basal-like bcs in a recent study investigating  <dig>  breast tumours  <cit>  and expression levels significantly correlated with their dna copy number in triple-negative bc cell lines and tumours for several of these genes  <cit> .
a recent study investigated genes on 5p <dig> -p <dig>  in more detail and showed that silencing of the overexpressed and amplified nuns <dig>  a myc target gene, reduced cell number in some bc cell lines  <cit> .
nuns <dig> expression has been found significantly increased in malignant tissues whereas it could only be found in testis in normal tissues, furthermore its role in stabilising the mitotic spindle and phosphorylation by aurora-b make it an interesting target for cancer diagnostics and molecular therapeutics.
CONCLUSIONS
taken together, transcriptional, genomic and epigenetic profiles of  <dig> bc cell lines, enriched for those representing triple-negative features, help to define cell lines that most closely capture individual examples of the heterogeneous characteristics within triple-negative bcs.
by cross-referencing different high-resolution datasets, we provide useful resources to further study transcriptional, as well as genetic and epigenetic modulation and inform the best selection of available in vitro models for the identification and validation of potential novel therapeutic targets relevant to triple-negative bcs.

 oestrogen-receptor negative breast cancer accounts for approximately 20% of all newly diagnosed breast malignancies. clinically, however, this group of bcs contains different subtypes and can be subdivided into either her2-positive or triple-negative bcs, defined by very low or absent immunohistochemical expression of er and progesterone receptor, and low expression and lack of amplification of her. triple-negative bcs account for 10-15% of all breast tumours and are mostly of high grade, have a high incidence of tp mutations, and show proliferative characteristics with a higher propensity to spread to visceral organs. sharing many of these phenotypic features with triple-negative bcs are breast tumours of the ‘intrinsic’ basal-like subtype. these tumours generally lack er and her expression and are molecularly characterised by the expression of genes associated with both basal epithelium and myoepithelium of the normal mammary gland. approximately, 80% of triple-negative bcs show features of basal-like bcs. while most triple-negative bcs show aggressive clinical behaviour and have very limited targeted therapies, they also encompass subgroups of cancers sensitive to chemotherapy and having a good prognosis. hence, continuous efforts to characterise this bc population have already identified several subgroups. one of the proposed groups comprise “claudin-low” tumours, which are characterised by gene expression profiles similar to those found in the so-called breast ‘cancer stem cell’ populations, while other subgroups were classified as having higher expression of the interferon-related or apocrine genes. bc cell lines are essential tools in bc research and have been widely used to elucidate bc biology and new therapies. since cell lines are easily propagated and genetically manipulated, extensive information about their transcriptome, genome and to a lesser extent epigenome has been produced. several studies have compared and integrated gene expression profiles and genomic alterations between primary breast tumours and bc cell lines, demonstrating that the heterogeneity found in primary bcs is to a certain extent recapitulated in the panel of commonly used bc cell lines. given the increasing knowledge of the diversity and complexity among bc subtypes it has also become evident that no individual cell line will recapitulate all aspects of the disease. here we interrogated genome-wide transcriptional profiles with genomic and epigenetic profiling in a collection of bc cell lines enriched for those of triple-negative phenotype. we have focused on gene signatures, underlying dna copy number aberrations and epigenetic events specifically associated with triple-negative bcs. by cataloguing these perturbations on a gene-centric basis we have extended the characterisation of these bc cell lines and offer valuable insights on their suitability in modelling certain features of this heterogeneous disease.  bc cell lines segregate into three groups based on their transcriptional profiles to investigate the molecular heterogeneity of triple-negative bc cell lines and their representativeness of triple-negative breast cancers, we used illumina humanwg-6v to survey the phenotypic and genotypic characteristics of seven er-negative mesenchymal bc cell lines and compared them with er-negative epithelial-like bc cell lines. five er-positive bc cell lines were also included in our dataset, to evaluate er-responsive transcriptional signatures in er-negative bc cell lines. in addition, two er-negative/ her2-positive epithelial bc cell lines were included, and were employed as comparators with other triple-negative bc cell lines. molecular pathological features of all bc cell lines are provided in additional file table s unsupervised hierarchical clustering of highly variable ensembl genes separated er-negative bc cell lines into three groups. one group, designated “cluster 1” included three er-negative/her2-positive bc cell lines, namely skbr sum and sum which clustered with er-positive/her2-negative and er-positive/her2-positive cell lines. “cluster 2” was uniformly composed of er-negative/her2-negative bc cell lines and was in complete concordance with the basal “b” bc cell line subtype described in previous bc cell line studies. “cluster 3” consisted of cell lines either having a triple-negative phenotype or showing amplification and higher abundance of her and egfr gene amplification. investigating the expression levels of known triple-negative bc-related genes demonstrated subtle differences within these three groups. while “cluster 3” cell lines expressed genes commonly found in the intrinsic gene list preferentially expressed in basal-like primary bc, “cluster 2” was characterised by a higher abundance of cav a marker for the basal-like phenotype of sporadic and hereditary breast cancers, and the lymphangiogenic factor vegfc, a potential therapeutic target for triple-negative bcs. other genes previously associated with triple-negative bcs such as c-met, cd and cav exhibited higher expression in both groups in comparison to “cluster 1” cell lines. representation of er-negative breast tumour-related gene signatures in bc cell lines given that the combined expression pattern of certain genes can uniquely characterise different bc subtypes or can be used as surrogate markers for pathway activation, we selected a compendium of gene signatures representing various features of triple-negative bcs. firstly, we investigated the representation of eleven gene signatures based on a “relative similarity score” and ranked the bc cell lines accordingly. overall “cluster 3” cell lines showed a good representation of gene signatures identified for “apocrine”, g tn.tumour” and high-grade breast carcinomas, while “cluster 2” cell lines exhibited high similarities with the “stroma”, “mammosphere” and “cd cd44” expression patterns. the resemblance to the remaining four gene signatures showed a less striking concordance with the gene expression defined clusters, and identified sum hcc sum and hcc cell lines as the best representatives for the “met”, “egfr”, “igf1” and “interferon” gene signatures, respectively. secondly, we examined the different er-negative bc subgroups associated with a prognostic outcome in the bc cell line transcriptomes based on centroid correlation classification. as shown in figure 2b, “cluster 3” cell lines had activation of the “cell cycle and cell proliferation pathway/immune response genes” and the “cell cycle and cell proliferation pathway” groups, both of which showed association to basal-like bcs in their study. in contrast, “cluster 1” cell lines displayed highest correlation to er-negative tumours of the steroid response group, supporting the outcome of our hierarchical clustering in which these cell lines group with the er-positive cell lines. lastly, to explore the representation of the molecular basal-like bc subtype within this bc cell line set, we performed nearest centroid classification using the class centroids from parker et al., sorlie et al., hu et al., prat et al. and guedj et al.,. as expected given the manner in which the “claudin.low” gene signature was originally established, “cluster 2” cell lines are representatives of the described “claudin.low” subtype. the recently published cit classification assigned all “cluster 2” and “cluster 3” cell lines to the basall group with the exception of hs578t which was assigned to the “luminala” subtype. interestingly, in “cluster 1”, er-negative/her2-positive cell lines skbr sum and sum were labelled as mapo groups, in agreement with er-negative/her2-positive breast carcinomas in this molecular subtype. pam classification assigned all “cluster and 3” bc cell lines except hs578t and hcc to the basal-like bc subtype. assignments of cell lines into the basal-like subtype with class centroids obtained from sorlie and hu were in good agreement with those obtained with pam however only out cell lines were consistently classified as of luminal a, luminal b or her by all methods. to determine the agreement of these three centroid classifications, we used the free-marginal kappa statistics of brennan and prediger and saw substantial agreement in the classification of the basal-like, luminal a and her subtypes, in agreement with the results of previous studies. copy number aberrations and associated gene expression changes in bc cell lines represent those observed in triple-negative bcs to identify bc cell lines harbouring copy number aberrations specific for triple-negative bc, we performed acgh using a 32k tiling path array platform and surveyed their genomic changes. by grouping the bc cell lines based on the three expression clusters, “cluster 2” cell lines displayed significantly less high-level amplifications and deletions compared with the other two clusters. next, we retrieved cnas identified in our previous study on triple-negative bcs, analysed on the same genomic platform. overall the frequency of gains and losses seen in triple-negative bcs was more similar to “cluster 3” than to “cluster 2” and “cluster 1” cell lines. recurrent amplification seen in primary triple-negative bcs were recapitulated in at least one bc cell line. the most highly recurrent triple-negative bc-specific amplicons in bc cell lines were on 5p -p, followed by 9p - and 7q found in “cluster 3” cell lines and “cluster 3” cell lines, respectively. given that these regions are also characterised by common polymorphisms, genes such as jak, nuns or limk previously associated with breast cancers, and gained preferentially in basal-like breast cancers might validate that these regions contain genes providing a selective advantage for triple-negative bcs. a comprehensive integration of genes lost or gained in the breast cancer study is provided in additional file table s amplifications on 3q24-q, 3q -q, 5p -p, 7p and 9p were observed only in ”cluster 3” cell lines, and the 13q -q amplicon only in “cluster 2” cell lines, illustrating a good representation of triple-negative bc-specific cnas in er-negative cell lines, with some of them having a higher prevalence in one than the other expression defined group. we and others have previously demonstrated that the expression levels of certain genes located in triple-negative bcs specific cnas is copy number dependent. to investigate if these dependencies are also recapitulated in bc cell lines, we integrated expression data with cbs-smoothed acgh profiles of each bc cell lines. using pearson’s correlation, genes showed significantly correlation between their expression and dna copy number levels. this set encompassed / triple-negative bc copy-number dependent genes as determined by turner, and included genes such as transcriptional regulators, kinases, phosphatases and transmembrane receptors, as well as biomarkers for diagnosis, prognosis, disease progression and known drug targets. triple-negative bc–specific amplicons, recurrently amplified in our bc cell lines and harbouring genes with dna copy number-dependent expression levels are shown in figure among those were genes, such as jak nsun and nfib previously shown to have pathogenic roles, but also novel potential drivers e.g. ppapdc or ranbp which could be selectively required for the survival of cells harbouring those amplification. epigenetic influence on sub-type specific genes in er-negative bc cell lines the functional validity of the methylation pattern found on cpg islands in cultured cancer cell lines has been the matter of controversy. to study epigenetic patterns in bc cell lines, we produced genome-wide methylation profiles on illumina goldengate bead arrays. unsupervised hierarchical clustering using cpg probes, corresponding to genes, revealed a different grouping as it was observed when their expression profiles were clustered. to validate that the bc cell line specific methylation pattern can be also found in triple-negative bc, we retrieved methylation data of fresh-frozen bcs performed on the same microarray. due to the lack of her status information in their study, basal-like bcs were used as surrogates for triple-negative tumours and cpg islands with variable methylation levels were observed. of those cpg probes showed also changes in their methylation status in our bc cell lines. investigating the methylation state of these cpg islands in each bc cell line individually, demonstrated an overall good representation of ≥70% of hypo- and hyper- basal-like bc-specific methylation events specifically in er-negative bc cell lines. given that bc subtype specific expression has been suggested to be under epigenetic influence, we surveyed the methylation effect on gene expression in bc cell lines. integration of methylation and gene expression data resulted in cpg gene pairs and identified an inverse correlation between methylation and gene expression levels for pairs. performing bootstrap analysis by randomly sampling the bc cell lines, we showed that the number of significant association between gene expression and methylation was more than fold higher than expected. using these cpg gene pairs in a multiclass sam analysis revealed with specific methylation patterns over the three expression clusters, particularly distinguishing “cluster 2” cell lines from the others. as described previously, “cluster 2” cell lines exhibited among others, expression patterns similar to the “claudin.low” gene signature. fifteen genes of the “claudin.low” gene signature had cpg sites with varying methylation pattern over these bc cell lines which was significantly higher than expected by chance, whereby genes downregulated in “claudin.low” cancers according to “claudin.low” signature were methylated and vice-versa, such as “claudin.low” signature genes such as prss cldn and vamp were downregulated in “cluster 2” and their cpg islands were methylated, while genes like sparc or ddr had unmethylated cpg islands and showed higher abundance in these bc cell lines. by comparing those with epigenetic-regulated genes in breast cancers, we identified genes with the same concordant pattern. taken together, our analyses demonstrate that bc cell lines retain methylation–dependent gene expression patterns observed in basal-like bcs, and strengthen an epigenetic influence on some bc phenotypes that are retained in their equivalent model systems. discussion triple-negative bcs represent a heterogeneous group with diverse deregulation of biological pathways. here, we extended the molecular characterisation of er-negative bc cell lines based on their genetic, epigenetic and transcriptional profiles, and correlated these with a comprehensive compendium of gene signatures reflecting different features of er-negative bcs. initial cluster analysis of bc cell lines’ expression profiles resulted in three groups, two clusters encompassing purely er-negative bc cell lines, while one consisted of three er-negative and all er-positive bc cell lines. the first two cell line clusters were in good agreement with recent bc cell line studies. while “cluster 2” encompassed cell lines that were all represented in the basal “b” cluster of neve et al. and were assigned to the triple-negative mesenchymal phenotype by lehmann et al., most of the “cluster 3” cell lines were part of neve’s basal “a” cluster and part of the basal-like subtype according to lehmann et al.,. our “cluster 3” cell lines exhibited expression patterns found in transcriptional profiles of microdissected grade triple-negative breast tumours as well as grade versus grade breast carcinomas. hcc an er-negative/her2-negative cell line, was the top in vitro representative “cluster 3” cell line for the triple-negative phenotype of microdissected grade triple-negative breast tumours. the transcriptional profile of hcc also seemed very suitable in modelling the interferon, igf and met signalling pathways. bc cell lines with expression patterns most closely associated with the apocrine.basal subtype were not defined to one or the other cluster and hcc an er-negative/her2-positive cell line of “cluster 3” displayed the highest representation. these bcs were originally defined on the basis of their androgen receptor level and many of them harboured erbb amplifications. this is in agreement with our findings, whereby using a recently published bc classifier, named cit, three er-negative/her2-positive cell lines skbr sum and sum were classified to the mapo breast cancer subtype. in a study, mdamb sum cal and mfm showed expression patterns associated with androgen receptor signalling and were more sensitive to androgen receptor antagonist bicalutamide and an hsp inhibitor. while none of those cell lines were part of our study, bt and hcc bc cell lines used in our study and good representatives of the apocrine.basal subtype showed high sensitivity to hsp inhibitors in lehmann’s work. the claudin-low subtype has been described as bc entity, which is enriched for er-negative invasive ductal carcinomas, while displaying low levels of luminal differentiation markers and activation of pathways involved in epithelial-to-mesenchymal transition, stem cell-like features and the immune response. integration of gene expression with methylation data over bc cell lines revealed a group of cpg islands corresponding to genes within the claudin-low signature, showing an inverse correlation between their methylation and the genes expression in bc cell lines and bcs. our findings are in agreement with those from a recent report that led to the identification of a set of genes whose expression was epigenetically regulated and when used as a gene signature identified mesenchymal features in claudin-low breast tumours. furthermore, they postulated that a deviant methylation might reflect cell lineage commitment in agreement with our hypothesis of a contribution of an epigenetic regulation to the claudin-low subtype. aberrant dna methylation events have initially been thought to accumulate in a random fashion within cells in pre-malignant tissues, however, lately it has also been shown that de novo methylation has a predictable pattern, creating plasticity followed by commitment to alternative cell lineages. holm and colleagues proposed that bc subtypes might be driven by different epigenetic events and could reflect their different cellular origins. nevertheless, an alternative hypothesis might also be that the methylation patterns are a result from mutations in genes controlling the epigenetic landscape in breast cancer; thus further investigation is warranted to determine whether these distinctive methylation patterns are results of genetic aberration in epigenetic regulator genes and/or contribute to delineation of the differentiation hierarchy of claudin-low and other bc subtypes. we and others have recently shown that basal-like bcs are most likely derived from luminal progenitor cells. identifying in vitro models would enhance our understanding of these cell populations. interestingly, our cluster and gene signature analysis revealed er-responsive features for skbr sum and sum three er-negative/her2-positive cell lines. skbr cells are well known to have luminal bc characteristics. in contrast, the classification of sum and sum is controversial. while some bc cell line studies assigned them to basal-like cell lines, others supported our finding of sum within the er-positive cluster. sum although not included in this study, was classified as of luminal phenotype in other studies. common to both is the expression of luminal cytokeratins and as well as genes found in luminal progenitor cell population, more consistent with a luminal classification. although sum was found to highly express aldh a marker for the so-called bc stem cells, further investigations are necessary to ascertain whether sum and sum represent appropriate in vitro models for luminal intermediate progenitor populations. high-level amplifications are less likely to represent random aberrations and often encompass genes driving the development or maintenance of tumour growth. three-quarters of triple-negative bcs harbour at least one amplicon, however, their recurrence rates are lower than those of high-level cnas found in er-positive/ her2-negative and her2-positive bc subtypes. here, we demonstrated that triple-negative bc-specific amplicons are recapitulated in er-negative bc cell lines and that some of them are associated with higher frequencies either to ”cluster 2” or “cluster 3” expression clusters. for example, the region on 5p -p was found to be recurrently amplified in 5/ and 10/ triple-negative bcs, was present in six ”cluster 3” but only in one ”cluster 2” cell lines. notably, these genomic sites map to regions of common germline copy number polymorphism and the functional consequences of their increased dna levels require further validation. nevertheless, several genes located within these amplified regions were found gained with a higher frequency in basal-like bcs in a recent study investigating breast tumours and expression levels significantly correlated with their dna copy number in triple-negative bc cell lines and tumours for several of these genes. a recent study investigated genes on 5p -p in more detail and showed that silencing of the overexpressed and amplified nuns a myc target gene, reduced cell number in some bc cell lines. nuns expression has been found significantly increased in malignant tissues whereas it could only be found in testis in normal tissues, furthermore its role in stabilising the mitotic spindle and phosphorylation by aurora-b make it an interesting target for cancer diagnostics and molecular therapeutics.  taken together, transcriptional, genomic and epigenetic profiles of bc cell lines, enriched for those representing triple-negative features, help to define cell lines that most closely capture individual examples of the heterogeneous characteristics within triple-negative bcs. by cross-referencing different high-resolution datasets, we provide useful resources to further study transcriptional, as well as genetic and epigenetic modulation and inform the best selection of available in vitro models for the identification and validation of potential novel therapeutic targets relevant to triple-negative bcs. 
target validation studies for all bc subtypes have largely employed established bc cell lines, which have proven to be effective tools for drug discovery. given the lines of evidence suggesting that bc cell lines are effective tools for drug discovery, we assessed the similarities between triple-negative bcs and cell lines, to identify in vitro representatives, modelling the diversity within this bc subtype. bc cell lines, enriched for those lacking er, pr and her expression, were subjected to transcriptomic, genomic and epigenomic profiling analyses and comparisons were made to existing knowledge of corresponding perturbations in triple-negative bcs. transcriptional analysis segregated er-negative bc cell lines into three groups, displaying distinctive abundances for genes involved in epithelial-mesenchymal transition, apocrine and high-grade carcinomas. combined methylation and gene expression analyses revealed a subset of genes characteristic of the claudin-low bc subtype, exhibiting epigenetic-regulated gene expression in bc cell lines and tumours, suggesting that methylation patterns are likely to underpin subtype-specificity. here, we provide a comprehensive analysis of triple-negative bc features on several molecular levels in bc cell lines, thereby creating an in-depth resource to access the suitability of individual lines as experimental models for studying bc tumour biology, biomarkers and possible therapeutic targets in the context of preclinical target validation. dna copy number aberrations of triple-negative bcs were well represented in cell lines and genes with coordinately altered gene expression showed similar patterns in tumours and cell lines. methylation events in triple-negative bcs were mostly retained in epigenomes of cell lines. triple-negative breast cancers represent a heterogeneous subtype of bcs, generally associated with an aggressive clinical course and where targeted therapies are currently limited. microarraygene expression profilingcomparative genomic hybridisationmethylation arraystriple negativebreast cancer
 triple-negative breast cancers represent a heterogeneous subtype of bcs, generally associated with an aggressive clinical course and where targeted therapies are currently limited. target validation studies for all bc subtypes have largely employed established bc cell lines, which have proven to be effective tools for drug discovery.  given the lines of evidence suggesting that bc cell lines are effective tools for drug discovery, we assessed the similarities between triple-negative bcs and cell lines, to identify in vitro representatives, modelling the diversity within this bc subtype. bc cell lines, enriched for those lacking er, pr and her expression, were subjected to transcriptomic, genomic and epigenomic profiling analyses and comparisons were made to existing knowledge of corresponding perturbations in triple-negative bcs. transcriptional analysis segregated er-negative bc cell lines into three groups, displaying distinctive abundances for genes involved in epithelial-mesenchymal transition, apocrine and high-grade carcinomas. dna copy number aberrations of triple-negative bcs were well represented in cell lines and genes with coordinately altered gene expression showed similar patterns in tumours and cell lines. methylation events in triple-negative bcs were mostly retained in epigenomes of cell lines. combined methylation and gene expression analyses revealed a subset of genes characteristic of the claudin-low bc subtype, exhibiting epigenetic-regulated gene expression in bc cell lines and tumours, suggesting that methylation patterns are likely to underpin subtype-specificity.  here, we provide a comprehensive analysis of triple-negative bc features on several molecular levels in bc cell lines, thereby creating an in-depth resource to access the suitability of individual lines as experimental models for studying bc tumour biology, biomarkers and possible therapeutic targets in the context of preclinical target validation. microarraygene expression profilingcomparative genomic hybridisationmethylation arraystriple negativebreast cancer 