BACKGROUND
breast cancer is a heterogeneous disease that comprises tumour subgroups with substantial differences in biology and clinical behaviour.
classification methods based on the expression of intrinsic genes  <cit>  and on the histological assessment of oestrogen-  and progesterone-  receptor and human epidermal growth factor receptor  <dig> , have revealed the existence of different subgroups with diverse clinical outcomes and responses to treatment.
among these, er-negative and triple-negative breast cancers  are types of aggressive tumours that account for approximately 30% and 15% of breast cancers, respectively  <cit>  and are known to have a poorer prognosis than most er-positive types.
although various multi-gene prognostic markers have been proposed for the prediction of their clinical outcome, the reliable identification of the small group of patients with er-negative tumours who have a more favourable prognosis still represents an open challenge .
mirnas are small non-coding rna molecules regulating gene expression both at the transcriptional and translational levels.
since  <dig>  when mirna deregulation was first described in breast cancer  <cit> , many studies have demonstrated a role for mirnas in the modulation of oncogenic pathways and their potential as prognostic and/or subtype-specific diagnostic biomarkers .
further interest in mirnas has stemmed from their demonstrated suitability for analysis in formalin–fixed, paraffin embedded  tumour tissues  <cit> .
until recently, only in a limited number of studies have mirnas been analysed in the context of transcriptional and genomic profiles obtained from the same breast tumour samples and integrated with clinical-pathological information  <cit> .
even more limited information exists when the focus is restricted to selected tumour sub-cohorts, such as triple-negative breast tumours.
thus, we carried out comprehensive mirna, mrna and dna profiling in a well-annotated cohort of invasive breast cancers , concentrating on triple-negative tumours .
a general overview of the rationale of the study is illustrated in figure  <dig>  integrative genomic data analysis, along with clinical and pathological information allowed us to identify prognostic and subtype-specific mirnas , to elucidate whether they were affected by dna genomic aberrations  and to monitor their associative and/or functional relationships with the activity of biological pathways.
for the latter point we adopted two independent strategies.
the first of these looked at the associative correlations between mirnas and the level of activation of a large compendium of pathways, inferred from gene-expression signatures .
the second strategy aimed to identify the subset of mirnas potentially playing an effect on the expression levels of their predicated targets and provided a comprehensive description of the pathways and gene sets most significantly influenced by these mirnas .
by using cox-regression analysis with distant metastases-free survival  and breast cancer specific survival  as clinical end points, we found  <dig> mirnas associated with prognosis in triple-negative tumours with an additional  <dig> when the analysis was extended to the larger group of all er-negative tumours.
when we investigated the pathways associated to these mirna, we found unfavourable prognostic mirnas to correlate with a broad spectrum of motility mechanisms involved in cell invasion and to growth factor-mediated signalling pathways.
to understand the role of mirnas in the establishment of tumour transcriptional phenotypes we also investigated mirna expression patterns across different intrinsic molecular subtypes, as defined by the pam <dig> classification method  <cit> , and identified  <dig> subtype-specific mirnas.
integrated mirna-mrna analyses showed that subtype-specific mirnas tend to be enriched for anti-correlated genes among their predicted targets.
this result represents an exception from the general lack of correlation that was observed between mirnas and predicted targets expression levels.
among subtype-specific mirnas, our analysis highlighted the role of mir17- <dig> and mir-106b- <dig>  two polycistronic mirna clusters known to play a key oncogenic role in various cancers  <cit> .
these two clusters appeared to be specifically over-expressed in basal-like tumours and their expression to be largely influenced by dna copy gains.
we also observed the anti-correlated predicted targets of mir17- <dig> and mir-106b- <dig> mirnas to be enriched for luminal specific sets of genes.
this suggests these mirnas contribute to the definition of the tumour transcriptional phenotype by influencing the expression levels of these genes.
interest in the mir17- <dig> and mir-106b- <dig> clusters was further strengthened by their inferred interference with known oncogenic processes, including epithelial-mesenchymal transition , pi3k/akt/mtor, myc and pten pathways.
RESULTS
a schematic representation of the integrated analytical workflow used to generate the presented results is illustrated in additional file 1: figure s <dig>
samples data and tumour classification
the study is based on the integrative analysis of mirna expression, gene expression , and snp-array based dna copy numbers  in a set of  <dig> invasive breast carcinomas extracted from  <dig> patients .
patients were treated at guy’s and st thomas’ hospitals, london, uk between  <dig> and  <dig> and had at least  <dig>  years follow-up.
of the  <dig> tumours analysed,  <dig> were immunohistochemically er-negative,  <dig> being also triple-negative .
molecular characteristics of tumour samples were analysed in association with clinical and pathological information .
in addition to the assignment to clinical subgroups based on er, pr and her <dig> status,  <dig> tumour samples were also assigned to the five intrinsic molecular subtypes  using the expression of predefined intrinsic gene lists according to the pam <dig> centroid-based classification method  <cit> .
in agreement with previous studies  <cit> , triple-negative breast cancers were found to correspond mostly with the basal-like tumours  while er-positive lesions corresponded to luminal a and b subtypes  .
mirna copy number and expression
mirnas are frequently located in regions of genomic instability  <cit> , and mirna expression changes have been found to be associated with chromosomal rearrangements in many tumours, including breast cancer  <cit> .
in order to gain a general view on the impact of dna aberrations on mirna expression in this cohort, spearman correlation coefficients between dna copy number of mirna loci  and mirna expression values were computed.
in addition, for each mirna dna locus identified as altered in any of the samples, we performed separate non-parametric wilcoxon rank sum tests to assess differences in expression between samples with losses and gains, compared to samples with no copy number alterations.
mirnas co-located with transposable elements  – given their uncertain genomic location were excluded from this analysis.
as a result we identified  <dig> mirnas showing statistically significant mirna-cn correlation, indicating an overall influence of genetic aberrations  on the expression of the mirnas .
17/ <dig> of these mirnas were identified to fall into breast cancer recurrent aberrant regions  <cit> .
of these, respectively  <dig> and  <dig> mirnas fall into regions of focal recurrent amplifications and focal recurrent deletions .
these results suggest the expression values of these mirnas to be frequently perturbed in breast cancers, as result of underlying dna aberrations.
we have then carried out an analysis to assess the likelihood that copy-number driven mirna could be co-amplified/co-deleted with key cancer related genes.
for each copy-number driven mirna, a region spanning  <dig> kilobases before and after its genomic location was isolated and oncogenes and tumor suppressor genes according to the "cancer genes db"  <cit>  were isolated.
this resulted in a list of co-amplified/co-deleted tumour suppressor and/or oncogenes for each mirna .
as examples, we found mir- <dig> and mir-106b to be co-amplified with pi3k and met and mir- <dig> to be co-amplified with myc, suggesting a  relationship between the expression levels of these mirnas and the activities of pi3k/akt, myc and met pathways.
expression analysis of mirna and candidate target transcripts
to assess the relationships between mirnas and their target genes, we carried out a correlation analysis between each mirna and the expression levels of their respective predicted transcripts.
the rationale for this analysis was based on three points: i) predicted targets are largely unreliable and therefore cannot be directly used to derive any sound biological observation ii) among all predicted mirna targets, the real ones - those transcriptionally degraded upon mirna binding - are anti-correlated to their cognate mirna  iii) mirnas showing an enrichment among their predicted targets for anti-correlated transcripts, are more likely to play a role in their control.
for each mirna, lists of candidate targets were extracted using six different prediction algorithms  and independent analyses were run on each list.
our data indicated the general lack of correlation between mirnas and predicted targeted mrnas, irrespective of the algorithm used for target prediction .
we focused then on the identification of individual mirnas deviating from this general behaviour, i.e.
for which a general anti-correlation with the expression levels of their predicted targeted mrnas could be detected.
we labelled these entities as mirnaapt  and devised a strategy for their identification based on the evaluation of their bias towards the enrichment for anti-correlated genes among their predicted targets .
by using stringent cut-offs we have identified a total of  <dig> mirnaapt .
mirnas associated with prognosis in er-negative and triple-negative breast cancers
cox-regression univariate analysis was carried out to identify mirnas whose expression was associated with clinical outcome.
analyses were run separately for the er-negative and the sub-set of triple-negative tumours, with respect to breast cancer specific survival  and distant metastases-free survival  clinical end points.
as result,  <dig> mirnas associated with prognosis were identified in er-negative tumours; of these  <dig> were also significant in the sub-set of triple-negative tumours  .
mirna-dna analyses showed mir-193a-3p to have the strongest association with dna copy numbers, with dna gains being associated with mirna over-expression.
prognostic mirnas tended to be sparsely distributed in the genome, with the exception of a cluster of mirnas located in close proximity to one another on band 14q <dig> , comprising mir-376b/mir-381/mir-409-5p/mir- <dig>  all linked to unfavourable prognosis.
we found, as have others, that this cluster is subject to frequent genomic deletions  <cit> , causing decrease in expression of mir-376b and mir- <dig> .
when univariate and multivariate analyses were run using histopathological information on its own , node positivity, tumour size and percentage of tumour lymphocytic infiltration emerged as statistically significant prognostic factors .
we then assessed how these covariates impacted mirna association with prognosis, by evaluating additive multivariate cox-regression models.
p-value distributions derived from different models were compared, showing an impact of the percentage of lymphocytic infiltration on the association of mirnas with prognosis .
mirnas were shown to be less informative with respect to prognosis when evaluated in multivariate models incorporating information on lymphocytic infiltration.
among the  <dig> mirnas collectively identified to be associated with prognosis alone, five retained their prognostic value when evaluated in models which included node positivity and tumour size  and only one  when lymphocytic infiltration was also considered, either in tnbc or in the wider group of er-negative samples  or both .
we have then analysed two independent mirna data sets: one from buffa et al., including  <dig> er-negative and  <dig> triple-negative breast cancer samples  <cit> ; the second from enerly et al, including  <dig> er-negative and  <dig> triple-negative samples  <cit> .
all analyses were run independently in the er-negative group and its subset of and triple-negative tumours.
analyses showed broad lack of reproducibility between the three sample cohorts with regard to mirna associations with prognosis .
furthermore, direct comparison between our results and the recently published results from the study of dvinge et al.
colleagues, again did not show overlap  <cit> .
as discussed more extensively below, the different sample size of the studies as well as differences in the cohort demographics  and the potential for differing representations of histopathological characteristics and biological subgroups within heterogeneous triple negative breast cancer, are all factors that might account for the general lack of reproducibility of analytical results with regard to the prognostic impact of mirnas.
finally, we have assessed how mirnas compare with mrnas with respect to association with bcss and dmfs.
we run cox-regression survival analysis on tnbc and er samples using gene expression and mirna data separately, without including histopathological information in the model.
when we compared the distributions of p-values, no significant differences between the two data sets emerged , indicating an overall comparable association of mirna and mrna to er-negative and tnbc patients prognosis.
pathways associated with prognostic mirnas in er-negative and triple-negative tumours
to gain insights into the biological role of the identified prognostic mirnas, we explored the relationships between their expression and a large compendium of pathways, biological processes and molecular functions represented by gene sets extracted from public  and commercial  databases.
the analysis was run using gene expression data to compute a "gene set score" for each gene set in each sample .
correlations between mirna expression levels and gene set scores across all tumour samples were calculated and used for unsupervised hierarchical clustering analysis.
in this way similarities between mirnas associated to the pathways represented by their respective gene sets could be highlighted.
as a result, a clear separation between unfavourable/favourable mirnas was obtained , indicating that their opposite prognostic values are reflected in the association with different classes of pathways .
many of the biological themes positively correlated with unfavourable prognostic mirnas are linked to cell motility, a critical step in the promotion of cancer invasion and metastasis, and to the broad spectrum of motility-related mechanisms such as cell adhesion, interaction with the extracellular matrix, cytoskeleton remodelling and control of membrane proteins through clathrin-mediated endocytosis.
others relate to intracellular signalling pathways, such as those activated by growth factors .
we also found a positive correlation with the renin-angiotensin system, a process reported to be involved in regulation of tumour angiogenesis in er-negative breast cancers  <cit> .
conversely, favourable prognostic mirnas were found to be associated with activities linked to proliferation such as enhanced cell division, inhibition of apoptosis, dna repair, protein and dna synthesis and metabolism.
subtype-specific mirnas
to understand the role of mirnas in the establishment of tumour transcriptional phenotypes we also investigated mirna expression patterns across different intrinsic molecular subtypes, as defined by the pam <dig> classification method  <cit> .
to this aim we carried out univariate anova analysis and identified  <dig> mirnas differentially expressed across the five intrinsic subtypes .
of these,  <dig> showed highest expression in basal-like,  <dig> in luminal tumours ,  <dig> in normal-like and  <dig> in her <dig> tumours .
one of them  was also prognostic in triple-negative tumours.
when we compared the list of specific subtype mirnas with that of the mirnas influenced by dna copy number changes we found  <dig> mirnas shared between the two groups, representing a statistically significant overlap .
this data indicates the tendency of specific subtype mirnas to be regulated at the genetic level, by an altered number of copies.
results on subtype-specific mirnas are consistent with those reported from independent studies  <cit>  .
notably, among the mirnas most up-regulated in basal-like tumours we observed members of the mir-17- <dig> cluster  and its paralog mir-106b- <dig> .
these two mirna clusters have been reported to be amplified and/or over-expressed in a variety of hematopoietic and solid tumours, including breast  <cit> , and are emerging as key modulators of various cancer –associated processes, including proliferation, apoptosis and angiogenesis .
analysis of gene sets potentially influenced by individual mirnaapt
we isolated for each mirnaapt the list of genes predicted to be targeted by prediction algorithms and, at the same time, being anti-correlated to the mirnaapt, assuming these lists to be enriched for real targets, as illustrated above.
running a gene set enrichment analysis on these targets-enriched lists we could then establish a link between mirnaapt and the gene sets potentially influenced by their action .
for the identification of the pathways and transcriptional signatures influenced by mirnaapt we used an adaptation of the approach described by creighton and colleagues  <cit> , based on gene set enrichment analysis of mirnas predicted targets through fisher’s exact test.
the algorithm was modified to use for each mirna only the list of anti-correlated predicted targets, and to run against a comprehensive compendium of published gene sets, representing published transcriptional signatures   <cit>  as well pathways, biological processes and molecular functions extracted from public  and commercial  that were significantly associated to one or more mirnaapt.
the complete set of results is reported in additional file 6: table s <dig>
the whole procedure was verified using external data on  <dig> mirnaapt, from an independent study by linsley et al.
<cit> .
for these mirnas, the effects on the predicted targets had been experimentally determined using mirna transfection experiments in in-vitro cancer cells, followed by gene expression analysis by microarrays.
in this way, they had derived for each mirna the list of down-regulated targeted transcripts in in-vitro cell lines.
we observed that these mrna gene sets were correctly predicted by our algorithm as being respectively influenced by the  <dig> mirnaapt tested.
in fact, for each of these mirnaapt, the experimentally validated list of its target genes showed up as the most significantly enriched gene set , thus providing direct evidence of the validity of the method.
moreover, the procedure has been extended based on mirna validated targets reported in the mirtarbase  <cit> .
for mirnas for which significant information on validated targets was available, such as let7-b, mir- <dig> and mir-29c, we could confirm many of our inferences on their targeted pathways.
examples are mir- <dig> association with pi3k-akt and tgf signalling pathways, let7-b association with pi3k and mir-29c association with pten pathways , discussed more extensively below.
transcriptional signatures and pathways potentially impacted by the action of subtype-specific mirnas
of the  <dig> subtype-specific mirnas,  <dig> were classified as mirnaapt suggesting a potential role of these mirnas in influencing the expression levels their targets .
among these are mirnas of the mir-17- <dig>  and mir-106b- <dig>  clusters, all up-regulated in the basal-like subtype of breast cancer.
these mirnas were associated to gene sets reported to be over-expressed in luminal and er-positive tumours or over-expressed in low-grade tumours, in independent studies .
in addition, mir-19b links to a gene set that is up-regulated in the her <dig> positive subtype of the disease.
we also observed the association with cancer related gene sets, such as the myc down-regulated gene set , as well as gene sets representing mtor and pten pathways .
other gene sets associated to mir-17- <dig> cluster include those related to tumour proliferation, such as the pdgf , tgf-β  and fgf  pathways, as well as gene sets involved in cell migration  and endocytosis .
furthermore, we observed the association of an epithelial-mesenchymal transition transcriptional signature by mir- <dig>  mir-19a/b and mir-106b.
mir-19b is also linked to elements of focal adhesion and endothelium, while mir-92a is involved with the regulation of cytoskeleton.
when we looked at luminal a and b specific mirnas, we found that let-7b/c and mir-29c link to gene sets that were down-regulated in luminal and er-positive tumours and up-regulated in basal-like and er-negative tumours.
cell cycle, proliferation and tumour grading gene sets are also found to be associated with let-7b/c, consistently with their reported role of as tumour suppressors, functioning as inhibitors of the cell cycle and regulators of apoptosis  <cit> .
interestingly, additional gene sets influenced by let-7b/c relate to the regulation of the immune system, in keeping with the proposed tumour suppressor role of let- <dig>  <cit> .
in order to assess the validity of our findings on the functional role of identified subtype-specific mirnaapt we have compared our results with experimental ones, derived from published independent in-vitro and in-vivo functional studies by others on mir-17- <dig> and mir-106b- <dig> mirnas .
many of these experiments confirmed our proposed relationships between mirnas and the gene sets and pathways influenced by their action.
these include experiments on mir-17- <dig> and mir-106b- <dig> clusters.
discussion
we present the results of the integrated analysis of mirna, mrna and dna data from a large breast cancer cohort strongly enriched for triple-negative types and extensively annotated with clinical and pathological information.
the work spans multiple lines of analysis, aiming to elucidate different aspects of mirna breast cancer biology.
what are the links between genomics aberrations, mirna expression levels and transcriptional down-regulation of target genes?
we found the expression levels of  <dig> mirnas to be statistically influenced by dna copy number changes.
this behaviour effects in particular sub-type specific mirnas and supports the notion that copy number alterations of mirnas account partly for mirna gene deregulation  <cit> .
of these  <dig>  respectively  <dig> and  <dig> mirnas fall into regions of focal recurrent amplifications and focal recurrent deletions, pointing towards a frequent expression perturbation of these mirnas as result of underlying dna aberrations.
as tumours harbour a great amount of transcriptional alterations it is difficult to distinguish "causes" - occurred upstream in the process of tumour formation - from downstream "effects" events.
the identification of genetically driven mirnas helps highlighting those mirnas that have been altered upstream in the chain of deregulation events, therefore being more likely to play a role in the establishment and maintenance of the tumour phenotype.
with respect to the relationships between mirna and cognate targets, recent evidence indicates that mirnas influence gene expression mainly by causing degradation of their target mrnas, and only to a smaller extent by inhibiting protein translation  <cit> .
however, in line with previous mirna-mrna analyses of breast tumours, as well as other settings  <cit> , our data showed a general lack of correlation between mirna expression and predicted mrna targets.
this is possibly due to the large presence of false positives in the list of predicted targets.
an additional explanation can be that the effects exerted by mirnas on their targets are limited to a subset of mirnas, rather than being broadly extended to all of them.
by means of a specific analytical workflow we identified  <dig> of these mirnas - that we named mirnaapt.
we then established functional links to the pathways and gene signatures on which mirnaapt are likely to exert a negative control.
do mirnas hold potential as prognostic markers in er-negative and triple-negative breast cancer?
we have identified  <dig> mirnas associated with clinical outcome in the set of all er-negative cancers;  <dig> of these were associated with prognosis also in the sub-group of triple-negative tumours.
we have then endeavoured to shed light on the pathways associated to the expression levels of each of these prognostic mirnas.
our analysis did not allow us to distinguish between associative and causal relationships.
however, the correlation of unfavourable prognostic mirnas with a number of cancer related pathways provides indirect  evidence of their association with the biology of the tumours.
in fact, among the pathways correlated with unfavourable mirnas, we found a recurrent emergence of cell motility and related mechanisms - processes that are known to facilitate the invasion of surrounding stromal tissues  <cit> .
other emerging themes associated with unfavourable mirnas were those related to tgf-β and egfr intracellular signalling pathways – known to play a pivotal role in metastasis  <cit>  – and to the stromal receptor ednrb – which is described as being involved in the acquisition of invasive potential of pre-malignant breast lesions  <cit> .
surprisingly, favourable mirnas were instead found to be positively associated with cell activities related to proliferation.
a possible explanation for this association is that tumour proliferation, as inferred from transcriptional data, could mirror the enhanced engagement and/or proliferation of immune cells , thus representing a protective factor.
a positive association between a proliferation metagene, a b cell metagene and good outcome in breast cancers has been observed by others in independent data sets .
further indications of the puzzling relationship between proliferation and patient survival in triple-negative breast cancers come also from recent studies, reporting the lack of association between standard markers of proliferation and prognosis in these types of tumours  <cit> .
when we compared our results with those obtained from independent data sets  <cit> , no overlaps were found.
these results point towards the elusive nature of mirnas when endeavours are made to validate their prognostic associations across independent studies as also previously highlighted  <cit> .
noticeably, lack of consistency also emerged when the three external studies were compared between themselves.
beyond the different powers deriving from different sample sizes of the studies, other factors may account for the observed inter-study differences.
among these are the cohort demographics .
another element which might account for inter-study differences is the impact of histopathological characteristics on the association of mirnas expression with the clinical follow-up - in particular the level of lymphocytic infiltration, as we have observed in our data.
thus, reported associations of mirna expression levels with prognosis might mirror - at least in part - different degrees of tumour lymphocytic infiltration, more than mirna specific processes occurring in tumour epithelial cells.
in fact, even though stromal cells are only a minority of the whole tumour sample, mirnas from both epithelial and stromal compartments will have been profiled and contributed to associations observed.
a related confounding factor is the inherent heterogeneity of triple-negative breast cancer  <cit> , whose subclasses might be differentially represented across different patient data sets.
these aspects are particularly critical in consideration that the great majority of mirnas are only weakly associated with prognosis, and point towards the need to take them into account when molecular-prognostic studies are carried out.
further experimental efforts are needed to shed light on the functional role of each of these mirnas in the triple-negative breast cancer setting.
by complementing top-down statistical approaches with the acquisition of functional information it will be possible to understand the biological mechanisms underpinning the link of a given mirna to prognosis, and possibly allowing dissection of the reasons why this link can be lost in different patient cohorts.
the pathway correlation analysis we have carried out for the  <dig> prognosis associated mirnas represents an endeavour in this direction.
however, whilst internal cross-validation checks of our results with the results from pathway analysis together provide an indication of their statistical and biological significance, further functional validation is warranted.
how are mirnas expressed in different intrinsic tumour subtypes?
we found  <dig> mirnas differentially expressed across different intrinsic tumour subtypes,  <dig> and  <dig> respectively showing the highest expression in basal-like and luminal a and b tumours .
as anticipated from the known relationships between the different classification schemes and pathological tumour features, mirnas associated with the basal-like intrinsic subtype tend to be also over-expressed in triple-negative and high-grade tumours.
the comparison of our results with equivalent analyses run in independent studies  <cit>  showed a very good level of agreement, therefore confirming their validity .
of interest, the link between breast cancer pam <dig> subtypes and mirna expression was also recently reinforced by the extensive analysis run by the cancer genome atlas network   <cit> .
here the authors presented an integrated genomic analysis of  <dig> breast cancer samples, classified on the basis of mrna expression - using the same pam <dig> classification criteria we have used - as well as mirna expression.
they demonstrated a strong convergence between the mirna- and mrna -based classification systems, pointing towards the fact that the pam <dig> subtype classification is reflected, at the mirna level, into the subtype-specific expression of a large number of mirnas.
we found the expression levels of  <dig> of the  <dig> subtype specific mirnas to be significantly associated with dna copy number changes, indicating a large fraction of mirna expression differences between different molecular subtypes can be ascribed to upstream genetic tumour aberrations.
these include the mir-17- <dig> and the mir-106b- <dig> mirna clusters, highly up-regulated in the basal-like tumours in this series and reported to be amplified and over-expressed in various tumours.
these clusters also function as modulators of different cancer processes, including proliferation, apoptosis and angiogenesis .
what is the role of subtype-specific mirnas in the establishment of different tumour transcriptional phenotypes?
out of the total of  <dig> subtype-specific mirnas,  <dig> were also identified to function as mirnaapt, suggesting a tendency of subtype-specific mirnas to influence the expression levels of their targets.
these included the basal-like breast cancer specific clusters mir-17- <dig>  and mir-106b- <dig> , and the luminal breast cancer specific mir- <dig> , let-7b/c and mir-29c .
clusters mir-17- <dig> and mir-106b- <dig> were shown to impact on transcriptional signatures reported to be up-regulated in er-positive, luminal tumours or down-regulated in er-negative basal-like tumours .
these findings open up a new perspective on the interpretation of these signatures, supporting the hypothesis that they can be significantly influenced by the action of mi-17- <dig> and mir-106b- <dig> clusters.
by also taking in consideration the mirna-dna and mirna-mrna associations previously discussed, a picture could be delineated in which genomic aberrations could contribute to the establishment of the transcriptional phenotype of basal-like tumours by altering the expression levels of mir-17- <dig> and mir-106b- <dig> mirnas  which, in turn, decrease the transcript levels of luminal-specific gene signatures.
it should be noted that the detected dna losses of the mir-17- <dig> region seem to affect the expression of the mirna cluster only to a limited, and not statistically significant, degree .
conversely, luminal a specific mirnas  were found to target gene sets down-regulated in luminal and er-positive tumours.
what is the role of subtype-specific mirnas in the regulation of oncogenic pathways?
we have identified and described a large number of cancer-related pathways potentially influenced by the action of mirnaapt.
these include the myc, mtor, tgf-β and pten and akt pathways as well as a transcriptional signature of emt.
our findings were supported by published experimental evidences of others , altogether pointing towards the plausibility of the inferences generated by our analytical procedure on the gene sets influenced by subtype-specific mirnaapt.
some of our findings could also be confirmed when only experimentally validated targets were used for the analysis, as extracted from the mirtarbase database  <cit> .
examples are mir- <dig> association with pi3k-akt and tgf signalling pathways and let7-b association with pi3k and mir-29c association with pten pathways .
analyses of cancer genes co-amplified/co-deleted with mirnas indicate that some of the identified links between these two mirnas and oncogenic pathways could be due to associative rather causal relationships.
examples are mir- <dig> and mir-106b, co-amplified with pi3k and met and mir- <dig> co-amplified with myc.
whilst a conclusive proof of our analytical findings will have to await functional experiments in basal-like breast cancer models, we believe these pieces of evidence provide indication of the overall validity of our analyses.
CONCLUSIONS
in this study we analysed mirna, mrna and dna data integrated with pathological and clinical information in a large and well-characterised cohort of triple-negative breast cancers.
the results presented advance current knowledge of the role of mirnas as modulators of oncogenic processes in these types of tumours, shedding light on their function in the control of subtype-specific transcriptional signatures and potential as biomarkers.
moreover, this study provides a conceptual framework, as well as integrative methods and system-level results to dissect the relationships between dna genomic aberrations and down-stream perturbations of mirnas, target genes and biological pathways.

 breast cancer is a heterogeneous disease that comprises tumour subgroups with substantial differences in biology and clinical behaviour. classification methods based on the expression of intrinsic genes and on the histological assessment of oestrogen- and progesterone- receptor and human epidermal growth factor receptor, have revealed the existence of different subgroups with diverse clinical outcomes and responses to treatment. among these, er-negative and triple-negative breast cancers are types of aggressive tumours that account for approximately 30% and 15% of breast cancers, respectively and are known to have a poorer prognosis than most er-positive types. although various multi-gene prognostic markers have been proposed for the prediction of their clinical outcome, the reliable identification of the small group of patients with er-negative tumours who have a more favourable prognosis still represents an open challenge. mirnas are small non-coding rna molecules regulating gene expression both at the transcriptional and translational levels. since when mirna deregulation was first described in breast cancer, many studies have demonstrated a role for mirnas in the modulation of oncogenic pathways and their potential as prognostic and/or subtype-specific diagnostic biomarkers. further interest in mirnas has stemmed from their demonstrated suitability for analysis in formalin–fixed, paraffin embedded tumour tissues. until recently, only in a limited number of studies have mirnas been analysed in the context of transcriptional and genomic profiles obtained from the same breast tumour samples and integrated with clinical-pathological information. even more limited information exists when the focus is restricted to selected tumour sub-cohorts, such as triple-negative breast tumours. thus, we carried out comprehensive mirna, mrna and dna profiling in a well-annotated cohort of invasive breast cancers, concentrating on triple-negative tumours. a general overview of the rationale of the study is illustrated in figure  integrative genomic data analysis, along with clinical and pathological information allowed us to identify prognostic and subtype-specific mirnas, to elucidate whether they were affected by dna genomic aberrations and to monitor their associative and/or functional relationships with the activity of biological pathways. for the latter point we adopted two independent strategies. the first of these looked at the associative correlations between mirnas and the level of activation of a large compendium of pathways, inferred from gene-expression signatures. the second strategy aimed to identify the subset of mirnas potentially playing an effect on the expression levels of their predicated targets and provided a comprehensive description of the pathways and gene sets most significantly influenced by these mirnas. by using cox-regression analysis with distant metastases-free survival and breast cancer specific survival as clinical end points, we found mirnas associated with prognosis in triple-negative tumours with an additional when the analysis was extended to the larger group of all er-negative tumours. when we investigated the pathways associated to these mirna, we found unfavourable prognostic mirnas to correlate with a broad spectrum of motility mechanisms involved in cell invasion and to growth factor-mediated signalling pathways. to understand the role of mirnas in the establishment of tumour transcriptional phenotypes we also investigated mirna expression patterns across different intrinsic molecular subtypes, as defined by the pam classification method, and identified subtype-specific mirnas. integrated mirna-mrna analyses showed that subtype-specific mirnas tend to be enriched for anti-correlated genes among their predicted targets. this result represents an exception from the general lack of correlation that was observed between mirnas and predicted targets expression levels. among subtype-specific mirnas, our analysis highlighted the role of mir17- and mir-106b- two polycistronic mirna clusters known to play a key oncogenic role in various cancers. these two clusters appeared to be specifically over-expressed in basal-like tumours and their expression to be largely influenced by dna copy gains. we also observed the anti-correlated predicted targets of mir17- and mir-106b- mirnas to be enriched for luminal specific sets of genes. this suggests these mirnas contribute to the definition of the tumour transcriptional phenotype by influencing the expression levels of these genes. interest in the mir17- and mir-106b- clusters was further strengthened by their inferred interference with known oncogenic processes, including epithelial-mesenchymal transition, pi3k/akt/mtor, myc and pten pathways.  a schematic representation of the integrated analytical workflow used to generate the presented results is illustrated in additional file 1: figure s samples data and tumour classification the study is based on the integrative analysis of mirna expression, gene expression, and snp-array based dna copy numbers in a set of invasive breast carcinomas extracted from patients. patients were treated at guy’s and st thomas’ hospitals, london, uk between and and had at least  years follow-up. of the tumours analysed, were immunohistochemically er-negative, being also triple-negative. molecular characteristics of tumour samples were analysed in association with clinical and pathological information. in addition to the assignment to clinical subgroups based on er, pr and her status, tumour samples were also assigned to the five intrinsic molecular subtypes using the expression of predefined intrinsic gene lists according to the pam centroid-based classification method. in agreement with previous studies, triple-negative breast cancers were found to correspond mostly with the basal-like tumours while er-positive lesions corresponded to luminal a and b subtypes. mirna copy number and expression mirnas are frequently located in regions of genomic instability, and mirna expression changes have been found to be associated with chromosomal rearrangements in many tumours, including breast cancer. in order to gain a general view on the impact of dna aberrations on mirna expression in this cohort, spearman correlation coefficients between dna copy number of mirna loci and mirna expression values were computed. in addition, for each mirna dna locus identified as altered in any of the samples, we performed separate non-parametric wilcoxon rank sum tests to assess differences in expression between samples with losses and gains, compared to samples with no copy number alterations. mirnas co-located with transposable elements – given their uncertain genomic location were excluded from this analysis. as a result we identified mirnas showing statistically significant mirna-cn correlation, indicating an overall influence of genetic aberrations on the expression of the mirnas. 17/ of these mirnas were identified to fall into breast cancer recurrent aberrant regions. of these, respectively and mirnas fall into regions of focal recurrent amplifications and focal recurrent deletions. these results suggest the expression values of these mirnas to be frequently perturbed in breast cancers, as result of underlying dna aberrations. we have then carried out an analysis to assess the likelihood that copy-number driven mirna could be co-amplified/co-deleted with key cancer related genes. for each copy-number driven mirna, a region spanning kilobases before and after its genomic location was isolated and oncogenes and tumor suppressor genes according to the "cancer genes db" were isolated. this resulted in a list of co-amplified/co-deleted tumour suppressor and/or oncogenes for each mirna. as examples, we found mir- and mir-106b to be co-amplified with pi3k and met and mir- to be co-amplified with myc, suggesting a relationship between the expression levels of these mirnas and the activities of pi3k/akt, myc and met pathways. expression analysis of mirna and candidate target transcripts to assess the relationships between mirnas and their target genes, we carried out a correlation analysis between each mirna and the expression levels of their respective predicted transcripts. the rationale for this analysis was based on three points: i) predicted targets are largely unreliable and therefore cannot be directly used to derive any sound biological observation ii) among all predicted mirna targets, the real ones - those transcriptionally degraded upon mirna binding - are anti-correlated to their cognate mirna iii) mirnas showing an enrichment among their predicted targets for anti-correlated transcripts, are more likely to play a role in their control. for each mirna, lists of candidate targets were extracted using six different prediction algorithms and independent analyses were run on each list. our data indicated the general lack of correlation between mirnas and predicted targeted mrnas, irrespective of the algorithm used for target prediction. we focused then on the identification of individual mirnas deviating from this general behaviour, i.e. for which a general anti-correlation with the expression levels of their predicted targeted mrnas could be detected. we labelled these entities as mirnaapt and devised a strategy for their identification based on the evaluation of their bias towards the enrichment for anti-correlated genes among their predicted targets. by using stringent cut-offs we have identified a total of mirnaapt. mirnas associated with prognosis in er-negative and triple-negative breast cancers cox-regression univariate analysis was carried out to identify mirnas whose expression was associated with clinical outcome. analyses were run separately for the er-negative and the sub-set of triple-negative tumours, with respect to breast cancer specific survival and distant metastases-free survival clinical end points. as result, mirnas associated with prognosis were identified in er-negative tumours; of these were also significant in the sub-set of triple-negative tumours. mirna-dna analyses showed mir-193a-3p to have the strongest association with dna copy numbers, with dna gains being associated with mirna over-expression. prognostic mirnas tended to be sparsely distributed in the genome, with the exception of a cluster of mirnas located in close proximity to one another on band 14q, comprising mir-376b/mir-381/mir-409-5p/mir- all linked to unfavourable prognosis. we found, as have others, that this cluster is subject to frequent genomic deletions, causing decrease in expression of mir-376b and mir-. when univariate and multivariate analyses were run using histopathological information on its own, node positivity, tumour size and percentage of tumour lymphocytic infiltration emerged as statistically significant prognostic factors. we then assessed how these covariates impacted mirna association with prognosis, by evaluating additive multivariate cox-regression models. p-value distributions derived from different models were compared, showing an impact of the percentage of lymphocytic infiltration on the association of mirnas with prognosis. mirnas were shown to be less informative with respect to prognosis when evaluated in multivariate models incorporating information on lymphocytic infiltration. among the mirnas collectively identified to be associated with prognosis alone, five retained their prognostic value when evaluated in models which included node positivity and tumour size and only one when lymphocytic infiltration was also considered, either in tnbc or in the wider group of er-negative samples or both. we have then analysed two independent mirna data sets: one from buffa et al., including er-negative and triple-negative breast cancer samples; the second from enerly et al, including er-negative and triple-negative samples. all analyses were run independently in the er-negative group and its subset of and triple-negative tumours. analyses showed broad lack of reproducibility between the three sample cohorts with regard to mirna associations with prognosis. furthermore, direct comparison between our results and the recently published results from the study of dvinge et al. colleagues, again did not show overlap. as discussed more extensively below, the different sample size of the studies as well as differences in the cohort demographics and the potential for differing representations of histopathological characteristics and biological subgroups within heterogeneous triple negative breast cancer, are all factors that might account for the general lack of reproducibility of analytical results with regard to the prognostic impact of mirnas. finally, we have assessed how mirnas compare with mrnas with respect to association with bcss and dmfs. we run cox-regression survival analysis on tnbc and er samples using gene expression and mirna data separately, without including histopathological information in the model. when we compared the distributions of p-values, no significant differences between the two data sets emerged, indicating an overall comparable association of mirna and mrna to er-negative and tnbc patients prognosis. pathways associated with prognostic mirnas in er-negative and triple-negative tumours to gain insights into the biological role of the identified prognostic mirnas, we explored the relationships between their expression and a large compendium of pathways, biological processes and molecular functions represented by gene sets extracted from public and commercial databases. the analysis was run using gene expression data to compute a "gene set score" for each gene set in each sample. correlations between mirna expression levels and gene set scores across all tumour samples were calculated and used for unsupervised hierarchical clustering analysis. in this way similarities between mirnas associated to the pathways represented by their respective gene sets could be highlighted. as a result, a clear separation between unfavourable/favourable mirnas was obtained, indicating that their opposite prognostic values are reflected in the association with different classes of pathways. many of the biological themes positively correlated with unfavourable prognostic mirnas are linked to cell motility, a critical step in the promotion of cancer invasion and metastasis, and to the broad spectrum of motility-related mechanisms such as cell adhesion, interaction with the extracellular matrix, cytoskeleton remodelling and control of membrane proteins through clathrin-mediated endocytosis. others relate to intracellular signalling pathways, such as those activated by growth factors. we also found a positive correlation with the renin-angiotensin system, a process reported to be involved in regulation of tumour angiogenesis in er-negative breast cancers. conversely, favourable prognostic mirnas were found to be associated with activities linked to proliferation such as enhanced cell division, inhibition of apoptosis, dna repair, protein and dna synthesis and metabolism. subtype-specific mirnas to understand the role of mirnas in the establishment of tumour transcriptional phenotypes we also investigated mirna expression patterns across different intrinsic molecular subtypes, as defined by the pam classification method. to this aim we carried out univariate anova analysis and identified mirnas differentially expressed across the five intrinsic subtypes. of these, showed highest expression in basal-like, in luminal tumours, in normal-like and in her tumours. one of them was also prognostic in triple-negative tumours. when we compared the list of specific subtype mirnas with that of the mirnas influenced by dna copy number changes we found mirnas shared between the two groups, representing a statistically significant overlap. this data indicates the tendency of specific subtype mirnas to be regulated at the genetic level, by an altered number of copies. results on subtype-specific mirnas are consistent with those reported from independent studies. notably, among the mirnas most up-regulated in basal-like tumours we observed members of the mir-17- cluster and its paralog mir-106b-. these two mirna clusters have been reported to be amplified and/or over-expressed in a variety of hematopoietic and solid tumours, including breast, and are emerging as key modulators of various cancer –associated processes, including proliferation, apoptosis and angiogenesis. analysis of gene sets potentially influenced by individual mirnaapt we isolated for each mirnaapt the list of genes predicted to be targeted by prediction algorithms and, at the same time, being anti-correlated to the mirnaapt, assuming these lists to be enriched for real targets, as illustrated above. running a gene set enrichment analysis on these targets-enriched lists we could then establish a link between mirnaapt and the gene sets potentially influenced by their action. for the identification of the pathways and transcriptional signatures influenced by mirnaapt we used an adaptation of the approach described by creighton and colleagues, based on gene set enrichment analysis of mirnas predicted targets through fisher’s exact test. the algorithm was modified to use for each mirna only the list of anti-correlated predicted targets, and to run against a comprehensive compendium of published gene sets, representing published transcriptional signatures as well pathways, biological processes and molecular functions extracted from public and commercial that were significantly associated to one or more mirnaapt. the complete set of results is reported in additional file 6: table s the whole procedure was verified using external data on mirnaapt, from an independent study by linsley et al.. for these mirnas, the effects on the predicted targets had been experimentally determined using mirna transfection experiments in in-vitro cancer cells, followed by gene expression analysis by microarrays. in this way, they had derived for each mirna the list of down-regulated targeted transcripts in in-vitro cell lines. we observed that these mrna gene sets were correctly predicted by our algorithm as being respectively influenced by the mirnaapt tested. in fact, for each of these mirnaapt, the experimentally validated list of its target genes showed up as the most significantly enriched gene set, thus providing direct evidence of the validity of the method. moreover, the procedure has been extended based on mirna validated targets reported in the mirtarbase. for mirnas for which significant information on validated targets was available, such as let7-b, mir- and mir-29c, we could confirm many of our inferences on their targeted pathways. examples are mir- association with pi3k-akt and tgf signalling pathways, let7-b association with pi3k and mir-29c association with pten pathways, discussed more extensively below. transcriptional signatures and pathways potentially impacted by the action of subtype-specific mirnas of the subtype-specific mirnas, were classified as mirnaapt suggesting a potential role of these mirnas in influencing the expression levels their targets. among these are mirnas of the mir-17- and mir-106b- clusters, all up-regulated in the basal-like subtype of breast cancer. these mirnas were associated to gene sets reported to be over-expressed in luminal and er-positive tumours or over-expressed in low-grade tumours, in independent studies. in addition, mir-19b links to a gene set that is up-regulated in the her positive subtype of the disease. we also observed the association with cancer related gene sets, such as the myc down-regulated gene set, as well as gene sets representing mtor and pten pathways. other gene sets associated to mir-17- cluster include those related to tumour proliferation, such as the pdgf, tgf-β and fgf pathways, as well as gene sets involved in cell migration and endocytosis. furthermore, we observed the association of an epithelial-mesenchymal transition transcriptional signature by mir- mir-19a/b and mir-106b. mir-19b is also linked to elements of focal adhesion and endothelium, while mir-92a is involved with the regulation of cytoskeleton. when we looked at luminal a and b specific mirnas, we found that let-7b/c and mir-29c link to gene sets that were down-regulated in luminal and er-positive tumours and up-regulated in basal-like and er-negative tumours. cell cycle, proliferation and tumour grading gene sets are also found to be associated with let-7b/c, consistently with their reported role of as tumour suppressors, functioning as inhibitors of the cell cycle and regulators of apoptosis. interestingly, additional gene sets influenced by let-7b/c relate to the regulation of the immune system, in keeping with the proposed tumour suppressor role of let-. in order to assess the validity of our findings on the functional role of identified subtype-specific mirnaapt we have compared our results with experimental ones, derived from published independent in-vitro and in-vivo functional studies by others on mir-17- and mir-106b- mirnas. many of these experiments confirmed our proposed relationships between mirnas and the gene sets and pathways influenced by their action. these include experiments on mir-17- and mir-106b- clusters. discussion we present the results of the integrated analysis of mirna, mrna and dna data from a large breast cancer cohort strongly enriched for triple-negative types and extensively annotated with clinical and pathological information. the work spans multiple lines of analysis, aiming to elucidate different aspects of mirna breast cancer biology. what are the links between genomics aberrations, mirna expression levels and transcriptional down-regulation of target genes? we found the expression levels of mirnas to be statistically influenced by dna copy number changes. this behaviour effects in particular sub-type specific mirnas and supports the notion that copy number alterations of mirnas account partly for mirna gene deregulation. of these respectively and mirnas fall into regions of focal recurrent amplifications and focal recurrent deletions, pointing towards a frequent expression perturbation of these mirnas as result of underlying dna aberrations. as tumours harbour a great amount of transcriptional alterations it is difficult to distinguish "causes" - occurred upstream in the process of tumour formation - from downstream "effects" events. the identification of genetically driven mirnas helps highlighting those mirnas that have been altered upstream in the chain of deregulation events, therefore being more likely to play a role in the establishment and maintenance of the tumour phenotype. with respect to the relationships between mirna and cognate targets, recent evidence indicates that mirnas influence gene expression mainly by causing degradation of their target mrnas, and only to a smaller extent by inhibiting protein translation. however, in line with previous mirna-mrna analyses of breast tumours, as well as other settings, our data showed a general lack of correlation between mirna expression and predicted mrna targets. this is possibly due to the large presence of false positives in the list of predicted targets. an additional explanation can be that the effects exerted by mirnas on their targets are limited to a subset of mirnas, rather than being broadly extended to all of them. by means of a specific analytical workflow we identified of these mirnas - that we named mirnaapt. we then established functional links to the pathways and gene signatures on which mirnaapt are likely to exert a negative control. do mirnas hold potential as prognostic markers in er-negative and triple-negative breast cancer? we have identified mirnas associated with clinical outcome in the set of all er-negative cancers; of these were associated with prognosis also in the sub-group of triple-negative tumours. we have then endeavoured to shed light on the pathways associated to the expression levels of each of these prognostic mirnas. our analysis did not allow us to distinguish between associative and causal relationships. however, the correlation of unfavourable prognostic mirnas with a number of cancer related pathways provides indirect evidence of their association with the biology of the tumours. in fact, among the pathways correlated with unfavourable mirnas, we found a recurrent emergence of cell motility and related mechanisms - processes that are known to facilitate the invasion of surrounding stromal tissues. other emerging themes associated with unfavourable mirnas were those related to tgf-β and egfr intracellular signalling pathways – known to play a pivotal role in metastasis – and to the stromal receptor ednrb – which is described as being involved in the acquisition of invasive potential of pre-malignant breast lesions. surprisingly, favourable mirnas were instead found to be positively associated with cell activities related to proliferation. a possible explanation for this association is that tumour proliferation, as inferred from transcriptional data, could mirror the enhanced engagement and/or proliferation of immune cells, thus representing a protective factor. a positive association between a proliferation metagene, a b cell metagene and good outcome in breast cancers has been observed by others in independent data sets. further indications of the puzzling relationship between proliferation and patient survival in triple-negative breast cancers come also from recent studies, reporting the lack of association between standard markers of proliferation and prognosis in these types of tumours. when we compared our results with those obtained from independent data sets, no overlaps were found. these results point towards the elusive nature of mirnas when endeavours are made to validate their prognostic associations across independent studies as also previously highlighted. noticeably, lack of consistency also emerged when the three external studies were compared between themselves. beyond the different powers deriving from different sample sizes of the studies, other factors may account for the observed inter-study differences. among these are the cohort demographics. another element which might account for inter-study differences is the impact of histopathological characteristics on the association of mirnas expression with the clinical follow-up - in particular the level of lymphocytic infiltration, as we have observed in our data. thus, reported associations of mirna expression levels with prognosis might mirror - at least in part - different degrees of tumour lymphocytic infiltration, more than mirna specific processes occurring in tumour epithelial cells. in fact, even though stromal cells are only a minority of the whole tumour sample, mirnas from both epithelial and stromal compartments will have been profiled and contributed to associations observed. a related confounding factor is the inherent heterogeneity of triple-negative breast cancer, whose subclasses might be differentially represented across different patient data sets. these aspects are particularly critical in consideration that the great majority of mirnas are only weakly associated with prognosis, and point towards the need to take them into account when molecular-prognostic studies are carried out. further experimental efforts are needed to shed light on the functional role of each of these mirnas in the triple-negative breast cancer setting. by complementing top-down statistical approaches with the acquisition of functional information it will be possible to understand the biological mechanisms underpinning the link of a given mirna to prognosis, and possibly allowing dissection of the reasons why this link can be lost in different patient cohorts. the pathway correlation analysis we have carried out for the prognosis associated mirnas represents an endeavour in this direction. however, whilst internal cross-validation checks of our results with the results from pathway analysis together provide an indication of their statistical and biological significance, further functional validation is warranted. how are mirnas expressed in different intrinsic tumour subtypes? we found mirnas differentially expressed across different intrinsic tumour subtypes, and respectively showing the highest expression in basal-like and luminal a and b tumours. as anticipated from the known relationships between the different classification schemes and pathological tumour features, mirnas associated with the basal-like intrinsic subtype tend to be also over-expressed in triple-negative and high-grade tumours. the comparison of our results with equivalent analyses run in independent studies showed a very good level of agreement, therefore confirming their validity. of interest, the link between breast cancer pam subtypes and mirna expression was also recently reinforced by the extensive analysis run by the cancer genome atlas network. here the authors presented an integrated genomic analysis of breast cancer samples, classified on the basis of mrna expression - using the same pam classification criteria we have used - as well as mirna expression. they demonstrated a strong convergence between the mirna- and mrna -based classification systems, pointing towards the fact that the pam subtype classification is reflected, at the mirna level, into the subtype-specific expression of a large number of mirnas. we found the expression levels of of the subtype specific mirnas to be significantly associated with dna copy number changes, indicating a large fraction of mirna expression differences between different molecular subtypes can be ascribed to upstream genetic tumour aberrations. these include the mir-17- and the mir-106b- mirna clusters, highly up-regulated in the basal-like tumours in this series and reported to be amplified and over-expressed in various tumours. these clusters also function as modulators of different cancer processes, including proliferation, apoptosis and angiogenesis. what is the role of subtype-specific mirnas in the establishment of different tumour transcriptional phenotypes? out of the total of subtype-specific mirnas, were also identified to function as mirnaapt, suggesting a tendency of subtype-specific mirnas to influence the expression levels of their targets. these included the basal-like breast cancer specific clusters mir-17- and mir-106b-, and the luminal breast cancer specific mir-, let-7b/c and mir-29c. clusters mir-17- and mir-106b- were shown to impact on transcriptional signatures reported to be up-regulated in er-positive, luminal tumours or down-regulated in er-negative basal-like tumours. these findings open up a new perspective on the interpretation of these signatures, supporting the hypothesis that they can be significantly influenced by the action of mi-17- and mir-106b- clusters. by also taking in consideration the mirna-dna and mirna-mrna associations previously discussed, a picture could be delineated in which genomic aberrations could contribute to the establishment of the transcriptional phenotype of basal-like tumours by altering the expression levels of mir-17- and mir-106b- mirnas which, in turn, decrease the transcript levels of luminal-specific gene signatures. it should be noted that the detected dna losses of the mir-17- region seem to affect the expression of the mirna cluster only to a limited, and not statistically significant, degree. conversely, luminal a specific mirnas were found to target gene sets down-regulated in luminal and er-positive tumours. what is the role of subtype-specific mirnas in the regulation of oncogenic pathways? we have identified and described a large number of cancer-related pathways potentially influenced by the action of mirnaapt. these include the myc, mtor, tgf-β and pten and akt pathways as well as a transcriptional signature of emt. our findings were supported by published experimental evidences of others, altogether pointing towards the plausibility of the inferences generated by our analytical procedure on the gene sets influenced by subtype-specific mirnaapt. some of our findings could also be confirmed when only experimentally validated targets were used for the analysis, as extracted from the mirtarbase database. examples are mir- association with pi3k-akt and tgf signalling pathways and let7-b association with pi3k and mir-29c association with pten pathways. analyses of cancer genes co-amplified/co-deleted with mirnas indicate that some of the identified links between these two mirnas and oncogenic pathways could be due to associative rather causal relationships. examples are mir- and mir-106b, co-amplified with pi3k and met and mir- co-amplified with myc. whilst a conclusive proof of our analytical findings will have to await functional experiments in basal-like breast cancer models, we believe these pieces of evidence provide indication of the overall validity of our analyses.  in this study we analysed mirna, mrna and dna data integrated with pathological and clinical information in a large and well-characterised cohort of triple-negative breast cancers. the results presented advance current knowledge of the role of mirnas as modulators of oncogenic processes in these types of tumours, shedding light on their function in the control of subtype-specific transcriptional signatures and potential as biomarkers. moreover, this study provides a conceptual framework, as well as integrative methods and system-level results to dissect the relationships between dna genomic aberrations and down-stream perturbations of mirnas, target genes and biological pathways. 
this study analyses previously unreported mirna, mrna and dna data and integrates these with pathological and clinical information, from a well-annotated cohort of breast cancers enriched for triple-negative subtypes. it provides a conceptual framework, as well as integrative methods and system-level results and contributes to elucidate the role of mirnas as biomarkers and modulators of oncogenic processes in these types of tumours. we showed that their basal-like subtype specific up-regulation is influenced by increased dna copy number and contributes to the transcriptional phenotype as well as the activation of oncogenic pathways in basal-like tumours. mirnas linked to an unfavourable prognosis were associated with a broad spectrum of motility mechanisms involved in the invasion of stromal tissues, such as cell-adhesion, growth factor-mediated signalling pathways, interaction with the extracellular matrix and cytoskeleton remodelling. this study focuses on the analysis of mirnas expression data in a cohort of well characterised breast cancer samples composed primarily of triple-negative tumours with associated genome-wide dna and mrna data, extensive patient follow-up and pathological information. when we compared different intrinsic molecular subtypes we found mirnas that were specifically expressed in one or more intrinsic subtypes. we identified mirnas associated with prognosis in the triple-negative tumours and an additional when the analysis was extended to the set of all er-negative cases. integrated genomic analyses indicated these mirnas to be influenced by dna genomic aberrations and to have an overall influence on the expression levels of their predicted targets. among others, our analyses highlighted the role of mir-17- and mir-106b- two polycistronic mirna clusters with known oncogenic functions.
 this study focuses on the analysis of mirnas expression data in a cohort of well characterised breast cancer samples composed primarily of triple-negative tumours with associated genome-wide dna and mrna data, extensive patient follow-up and pathological information.  we identified mirnas associated with prognosis in the triple-negative tumours and an additional when the analysis was extended to the set of all er-negative cases. mirnas linked to an unfavourable prognosis were associated with a broad spectrum of motility mechanisms involved in the invasion of stromal tissues, such as cell-adhesion, growth factor-mediated signalling pathways, interaction with the extracellular matrix and cytoskeleton remodelling. when we compared different intrinsic molecular subtypes we found mirnas that were specifically expressed in one or more intrinsic subtypes. integrated genomic analyses indicated these mirnas to be influenced by dna genomic aberrations and to have an overall influence on the expression levels of their predicted targets. among others, our analyses highlighted the role of mir-17- and mir-106b- two polycistronic mirna clusters with known oncogenic functions. we showed that their basal-like subtype specific up-regulation is influenced by increased dna copy number and contributes to the transcriptional phenotype as well as the activation of oncogenic pathways in basal-like tumours.  this study analyses previously unreported mirna, mrna and dna data and integrates these with pathological and clinical information, from a well-annotated cohort of breast cancers enriched for triple-negative subtypes. it provides a conceptual framework, as well as integrative methods and system-level results and contributes to elucidate the role of mirnas as biomarkers and modulators of oncogenic processes in these types of tumours. mirnasbreast cancerdata integrationpathway analysis 