BACKGROUND
epithelial ovarian cancer  is the second most common and the most deadly malignancy of the female reproductive tract.
serous, endometrioid, clear-cell, and mucinous ovarian cancers are the four most common histotypes  <cit> .
the majority of eocs are diagnosed at stage iii and iv when the tumor cells are spread in the peritoneum along with the presence of malignant ascites.
the serous histotype accounts for about 80% of eocs, and the majority show an inactivating mutation of the tumor suppressor gene tp <dig>  low malignant potential  serous eocs are thought to arise by the transformation of tumors of borderline malignancy, and activating mutations in members of the ras pathway  are found in the majority of these tumors  <cit> .
lmp eocs show a relatively high growth capacity, are usually not invasive but resistant to conventional chemotherapy  <cit> .
a number of studies suggest that factors related to the inflammation of the ovarian surface epithelium  such as ovulation, endometriosis, and pelvic inflammatory diseases are associated with an increased risk for eoc  <cit> .
the most important hypothesis regarding eoc carcinogenesis is the ovulation theory, which relates the risk of ovarian cancer to incessant ovulation.
recently, it has been hypothesized that high grade serous, endometrioid and clear cell ovarian cancers arise from the fallopian tube epithelium and share a common pathogenic mechanism, i.e., iron-induced oxidative stress derived from retrograde menstruation  <cit> .
both the incessant ovulation and oxido-reductive fallopian tube epithelial damage hypotheses have provided evidence that inflammatory responses induced under physiological conditions may foster the development of eoc.
in accordance with these hypotheses of ovarian tumorigenesis, a number of cyto/chemokines has been found at detectable levels in ascites from eoc patients  <cit> .
among those molecules, il <dig> is the cytokine that has been most frequently associated with eoc carcinogenesis and progression  <cit> .
preclinical evidence has shown that il <dig> enhances tumor cell survival and increases resistance to chemotherapy via jak/stat signaling in tumor cells  <cit>  and il <dig> receptor alpha trans-signaling on tumor endothelial cells  <cit> .
in addition, il <dig> has pro-angiogenic properties  <cit> , regulating immune cell infiltration, a stromal reaction, and the tumor-promoting actions of th <dig> lymphocytes  <cit> .
in patients with advanced disease, high plasma levels of il <dig> correlate with poor prognosis  <cit>  and elevated levels are also present in malignant ascites  <cit> .
treatment of eoc cells with the anti-il <dig> antibody  siltuximab has been shown to reduce constitutive cyto/chemokine production and inhibit il <dig> signaling, tumor growth, the tumor-associated macrophage infiltrate, and angiogenesis in il6–producing intraperitoneal ovarian cancer xenografts  <cit> .
il <dig> stimulates inflammatory cytokine production, tumor angiogenesis and the tumor macrophage infiltrate in ovarian cancer and these actions can also be inhibited by a neutralizing anti-il <dig> ab in clinical studies  <cit> .
however, further knowledge on il6-expressing eocs is needed to select patients who are possibly responsive to il6-dependent therapies.
we have recently found that il <dig> can be co-expressed together with plasminogen activator inhibitor - <dig>  encoded by serpine <dig>  in a subset of advanced stage serous eocs due to the activation of the ligand-dependent egfr/nfkb signaling cascade  <cit> .
ex vivo, using  <dig> eocs from advanced-stage patients with malignant ascites at surgery, we observed co-expression of egfr, il <dig>  and pai- <dig> in 57% of primary tumors and concomitant expression of both il <dig> and pai- <dig> in the corresponding ascites.
computational analysis on four publicly available data sets of eoc gene expression showed a correlation between the expression of the il <dig> and serpine <dig> genes in advanced stage eoc patients, which in one case was associated with shorter progression-free survival  <cit> .
these results further highlight the involvement of il <dig> in the progression of eoc.
herein, to give further insight in the biology of il6-expressing serous eoc we utilized a bioinformatics approach, described in the flowchart of figure  <dig>  to identify il <dig> co-regulated genes and signaling pathway/s in which they are involved.
first, we identified a list of genes representing a molecular signature for both advanced-stage and lmp serous eoc which recapitulate the so-called ‘early growth factor response’.
we also identified an il6-correlated signature of seven genes involved in vascular thrombosis specific for advanced-stage serous eocs.
RESULTS
il <dig> expression significantly correlates with a defined gene set in advanced stage serous eocs
pearson’s correlation analysis of seven data sets containing the expression profiles of  <dig> samples from serous eocs  was performed to identify genes whose expression was significantly correlated with il <dig> expression in each data set.
correlation scores of each gene pair were computed using the r program essentially as described  <cit> .
for genes represented by multiple probes in the same array format, the probe with the highest correlation to il <dig> in the data set with the highest number of patients was chosen and considered for the other data sets when present .
this analysis allowed the identification of genes whose expression positively correlated with il <dig> along the seven data sets with a p-value ≤ <dig>  and a pearson’s correlation coefficient  exceeding  <dig>  .
a further analysis across the seven data sets yielded  <dig> concordant correlated genes in at least four data sets .
of note,  <dig> of  <dig> genes were correlated in data set i  obtained with an affymetrix platform, and in data set vii  obtained with an agilent platform.
among the identified il6-correlated genes cxcl <dig>  hbegf, serpine <dig>  dusp <dig>  zfp <dig>  and ier <dig> were common to all data sets.
the correlation between il <dig> and hbegf, an egfr ligand, and serpine <dig>  encoding pai- <dig>  is in agreement with our previously published results on co-expression of il <dig> and pai- <dig> in high grade eocs due to egfr activation  <cit> .
the majority of genes are associated to the biological process ‘proliferation’  .
among the genes associated with proliferation, there were a number of growth factor early response genes .
the il6-correlated signature also included genes associated with ‘inflammation’ , and the remaining genes were associated with ‘cell cycle and apoptosis’, ‘metabolism’ and ‘migration and invasion’.
a biological functions were defined using genealacart tool  <cit> .
thus, we identified a gene signature of il <dig> correlated genes in serous eoc containing mainly proliferation-associated genes.
advanced stage eoc-specific il6-correlated gene signature functionally associated with control of cell morphology and cardiovascular disease
next, to determine whether the identified gene signature was specific for advanced stage eoc or could also be associated with lmp eoc, pearson’s correlation analysis to il <dig> was applied to gene expression data of lmp eocs reported in data sets i, ii, and iv .
the density plot of il <dig> intensities showed a similar trend of expression in advanced stage and lmp eocs .
the data obtained comparing advanced stage eocs and lmp eocs were similar in the three data sets, and were more reliable  in data set i. among the above identified advanced-stage eocs il6-correlated genes,  <dig> were also significantly correlated in lmp eocs, while  <dig> genes  were specific for advanced-stages only .
among these genes, il <dig> has already been associated with aggressiveness and progression of malignant eoc  <cit> , while the others have not previously been associated with eoc biology and clinical outcome.
to provide insight into the possible biological significance of the 40-gene signature, functional analysis of positively correlated genes  was carried out by ingenuity pathway analysis software   <cit> .
the top two functions , associated with the highest score network, were ‘cell death, cellular function and maintenance, hematological system development and function’ and ‘cell death, cellular development, cellular growth and proliferation’ .
when ipa analysis was performed on the seven-gene signature specific for advanced stage eocs, the top function, associated with the highest score network, was ‘cell morphology, cell function, cardiovascular disease’.
as shown in figure  <dig> and listed in additional file  <dig>  all seven genes are included in this network  together with genes already known to have a role in the progression of eocs such as vegf, the receptor tyrosine kinases egfr and her <dig>  and the pi3k complex  <cit> .
in addition to the input genes, it is noteworthy that il <dig> is not present in the identified networks , but when added manually to each network establishes a connection with some of the correlated genes .
the nfkb complex was included in networks n <dig> and n <dig> , highlighting its possible pivotal role in eoc progression.
it thus appears that two signatures are related to il <dig> in eocs: a 33-gene signature common to advanced stage and lmp eocs and associated to control of cell growth and death, while the 7-gene signature, associated only to advanced-stage eocs likely presenting nfkb transcriptional activation, might be a determinant of tumor aggressiveness, and may be associated with a pathway regulating vascular thrombosis.
an il6-correlated gene set recapitulates the early growth factor response
to give further insight in the biology of eocs expressing il <dig>  a gsea  <cit>  analysis was performed for each data set listed in table  <dig>  by the “use a gene  as phenotype” analysis, gsea first ranks the genes according to their correlation to il <dig>  it then determines whether a priori defined set of genes, in this instance those belonging to the c <dig> curated catalogue of functional gene sets, are randomly distributed throughout the gene list or primarily found at the top or bottom.
common significant gene sets obtained from gsea analysis of the two largest data sets  were selected and analyzed in the other datasets.
this yielded  <dig> significantly enriched gene sets for all datasets.
normalized enrichment scores and fdr values in the different datasets are listed in table  <dig>  a literature search was conducted to identify signaling pathways previously implicated in the progression of eocs and/or in epithelial–derived malignancies.
among the most significant gene sets, the bild_kras_oncogenic_signature  <cit>  which includes genes whose expression is induced by the activation of h-ras oncogene, was originally derived from the herein named data set iii and can be considered a positive control.
three additional gene sets, amit_egf_response_60_hela, amit_egf_response_120_hela  <cit>  and nagashima_nrg1_signaling_up  <cit> , were considered possible candidates of signaling pathways associated with eoc, and are associated with ‘growth factor response’.
these gene sets comprise early response genes, i.e.
the egr family members, and the negative feedback regulators of the growth factor signaling, i.e.
zfp <dig> and klf <dig>  the fifth selected gene set, named kim_wt1_target_up in some ways also recapitulates the growth factor response, since among wt <dig> target genes the egf family ligands ereg, areg and hbegf are included  <cit> .
nes normalized enrichment score, fdr false discovery rate.
furthermore, among the wt <dig> target genes serpine <dig> was also identified in the same study.
enrichment plots related to the above described gene-sets in data set i are shown in figure  <dig>  it is of note that il <dig> is not included in the selected gene sets  as well as other genes that are included in network  <dig> identified by analysis using ipa.
based on the results obtained by the above-described computational analysis and on our recent demonstration that il <dig> is up-modulated in eoc cells upon egf stimulation in time-dependent manner  <cit> , in vitro validation of  <dig> genes selected from the il6-correlated gene sets was performed with real time rt-pcr using total rna from egf-stimulated serous eoc cell lines .
the il <dig> was up-modulated in all eoc cells analyzed upon egf stimulation.
concordantly, 75%, 58%, and 75% of the gene transcripts were up-modulated in igrov <dig>  oaw <dig>  and skov <dig> cells, respectively .
among the correlated genes common to  <dig> data sets , cxcl <dig>  hbegf, serpine <dig> and dusp <dig> were increased in all three eoc cell lines analyzed.
additionally, nr4a <dig>  a correlated gene in  <dig> data sets, was up-modulated upon egf stimulation in all eoc cells.
thbd and klf <dig> transcripts, associated with ’cardiovascular disease’ by ipa analysis, were up-modulated in  <dig> of  <dig> egf-stimulated eoc cells.
in contrast, the mmp <dig> transcript, whose relevant protein is associated with invasion and tumor progression  <cit> , was not up-modulated in egf-stimulated eoc cells.
interestingly, in non-transformed ovary cells, named iose- htert <dig>  <cit> , although il <dig> was slightly up-modulated by egf stimulation, only 25% of the transcripts analyzed were up-modulated.
these data indicate that ligand-dependent egfr activation in serous eoc cells induces the transcription of genes correlated with il <dig> expression.
discussion
microarray technology has developed very rapidly, and it has become relatively easy to analyze the expression levels of thousands of genes within cancer cells.
however, genes do not act in isolation, but each acts in complexes and builds networks and activated pathways that ultimately give rise to a specific cell phenotype.
thus, the search of co-regulated genes applying bioinformatics approaches may spread light on the biology of a tumor and its development.
previously, by applying this kind of ‘in silico’ approach on gene expression profiles of ovarian and thyroid carcinomas  <cit>  and melanomas  <cit> , we have been able to identify novel signaling pathways activated in those tumors.
the present study, by applying similar bioinformatics tools, highlights possible novel signaling pathways activated in il6-expressing eocs.
among those, growth factor-dependent signaling was also experimentally validated in vitro in selected cellular eoc models.
first of all, pearson’s correlation analysis allowed the identification of genes co-regulated with il <dig> in aggressive eoc providing evidence that co-regulated genes can encode proteins involved in common signaling pathways.
to identify il6-coregulated genes we adopted thresholds which allowed to obtain a good balance among the statistical significance, the strength of the correlation and the biological reproducibility.
furthermore, we performed the analysis on  <dig> different data sets, containing the gene expression profiles of more than  <dig> eoc samples, obtained on different array platforms, to increase the robustness on the bioinformatics results.
we found a gene signature common to both advanced stage and lmp serous eocs, and another 7-gene signature specific for advanced stage eocs.
the integration of the results obtained by ipa and gsea, allowed us to determine that all eocs, independently of their aggressiveness, co-regulate il <dig> together with genes associated with cell growth and early growth factor response, arguing for the hypothesis of common mechanism/s of transformation.
only advanced-stage eocs appeared to be characterized by a scenario that involves genes such as fpr <dig>  klf <dig> and thbd, to date associated with thrombosis and cardiovascular disease, thus suggesting that this pathway contributes to the growth and/or the spread of this type of tumor.
our data indicate the existence of a biological interaction between il <dig> expression and that of the co-regulated genes as resulted upon ipa and gsea analyses.
on the other hand, since knockdown of il- <dig> by specific sirna did not affect the amount of the transcripts analyzed , the regulation of the expression of the identified genes appeared not directly dependent to that of il <dig>  although the il <dig> gene was not associated with the networks identified by ipa or with the gene sets selected by gsea, these results are in agreement with our previous observations in a subset of advanced stage eoc where ligand-dependent egfr activation induced nfkb-dependent transcription of il <dig> together with pai- <dig>  encoded by the serpine <dig> gene  <cit> .
nfkb also emerged to be a possible transcriptional regulator of  <dig> out of  <dig> genes according to the reported informations  <cit> , data which might further indicate that a growth factor-dependent nfkb signaling is activated in a subset of eoc.
it is noteworthy that il <dig> and  <dig> of the  <dig> correlated genes were found up-modulated upon  <dig> hr serum stimulation of quiescent keratinocytes  <cit> .
we can therefore argue that the activation of growth factor activated signaling can either directly or indirectly induce the expression of il <dig> and genes which likely play a role in the growth of eocs.
furthermore, this growth factor-induced signaling pathway induces positive regulators of cellular function that are in turn regulated by negative feedback regulators such as zfp <dig> and klf <dig>  <cit> .
the hbegf gene, encoding for an egfr ligand, was also highly significantly correlated in all seven data sets analyzed, indicating the prevalence of ligand-dependent egfr activation.
the regulation of growth factor signaling pathways by negative feedback is a universal mechanism for limiting the duration and intensity of signaling output.
while negative feedback is a key component of normal cellular signaling, its role in cancer cells is more complex.
indeed, the loss of some negative feedback regulators might contribute to tumor progression, but might also be expressed at considerably higher levels in oncogene-mutant tumors as observed in braf-mutated melanomas  <cit> .
interestingly, the presence of a feedback negative mechanism has also been associated with greater efficacy of growth factor receptor-targeted therapy  <cit> .
the fact that in eoc cells, with active egfr/nfkb/il <dig> signaling, egfr-targeted therapy was more effective might be due to the up-regulation of feedback negative regulators of growth factor signaling  <cit> .
taken together, these data suggest that the il6-associated signature might have a translational impact helping to select eoc patients who are likely responsive to egfr-targeted therapy.
experiments are now ongoing to verify this hypothesis.
nuclear expression of the wilm’s tumor suppressor is found in ose cells and in the majority of serous eocs  <cit> .
however, the corresponding gene, named wt <dig>  has been never associated with il <dig> gene expression.
wt <dig> is required for kidney development, and the report in which the relevant gene set has been derived particularly emphasized the finding that the genes encoding the egf family ligands ereg, areg, and hbegf may be transcriptionally regulated by wt <dig>  orchestrating a fine-tuning of the egf signaling pathway  <cit> .
altogether these observations support that the egf signaling pathway is pivotal in the biology of eoc.
the gene signature common to advanced stage serous and lmp eocs is not unexpected if one considers the theory that lmp eocs derive from serous low grade eoc with a borderline morphologic phenotype  <cit> .
however, if this is the case, advanced-stage and lmp tumors might share common genetic alterations that induce aberrant growth.
in addition, in vitro validation experiments performed on gene transcripts of non-transformed surface ovary cells argue for the notion that the signature associated with a growth factor response is not expressed and/or egf-dependent in normal ovary cells.
the bioinformatics approach also produced hypothesis-generating results.
the association of the 7-gene signature with advance stage eocs is novel.
at present only angiogenesis-related genes and proteins, such as vegf and its receptor, have a well documented role in eoc biology and are already well-exploited targets in the therapy of more aggressive eocs  <cit> .
our findings open new questions on the role of genes associated with thrombosis and cardiovascular disease in the progression of eocs.
it has been recently hypothesized that low-dose aspirin as antithrombotic therapy may inhibit progression rather than the induction of eoc  <cit> .
indeed, aspirin and selective cox inhibitors could reduce progression not only by inhibiting prostaglandin production, thus reducing inflammation, but also by negatively modulating thrombosis-associated genes.
therefore, the inhibition of both pathways synergistically might be an interesting approach to block the growth and dissemination of advanced stage eocs.
mmp <dig>  a gene of the 7-gene signature specific for malignant eocs and encoding the metallo-protease   <dig> was present in network  <dig> of ipa analysis, but was not in any of the gene sets selected by gsea analysis.
mmps are key molecules of tumor cell invasion, including eocs  <cit>  and, since the majority of samples were advanced stage eocs, mmp <dig> could be a new player in the dissemination of these tumors and experiments are now ongoing to test its presence and role in advanced stage eocs.
CONCLUSIONS
by applying a bioinformatics approach we identified genes co-regulated with il <dig> expression in clinically-relevant subtypes of eoc, their interactions in networks and pathways as well as their functional association to growth factor response.
il <dig> gene expression together with that of the correlated gene signature could help identifying eoc patient’s subgroups in which the identified signaling pathways might be biologically relevant during the progression of the disease and, in the long term, might represent new pharmacological targets.

 epithelial ovarian cancer is the second most common and the most deadly malignancy of the female reproductive tract. serous, endometrioid, clear-cell, and mucinous ovarian cancers are the four most common histotypes. the majority of eocs are diagnosed at stage iii and iv when the tumor cells are spread in the peritoneum along with the presence of malignant ascites. the serous histotype accounts for about 80% of eocs, and the majority show an inactivating mutation of the tumor suppressor gene tp low malignant potential serous eocs are thought to arise by the transformation of tumors of borderline malignancy, and activating mutations in members of the ras pathway are found in the majority of these tumors. lmp eocs show a relatively high growth capacity, are usually not invasive but resistant to conventional chemotherapy. a number of studies suggest that factors related to the inflammation of the ovarian surface epithelium such as ovulation, endometriosis, and pelvic inflammatory diseases are associated with an increased risk for eoc. the most important hypothesis regarding eoc carcinogenesis is the ovulation theory, which relates the risk of ovarian cancer to incessant ovulation. recently, it has been hypothesized that high grade serous, endometrioid and clear cell ovarian cancers arise from the fallopian tube epithelium and share a common pathogenic mechanism, i.e., iron-induced oxidative stress derived from retrograde menstruation. both the incessant ovulation and oxido-reductive fallopian tube epithelial damage hypotheses have provided evidence that inflammatory responses induced under physiological conditions may foster the development of eoc. in accordance with these hypotheses of ovarian tumorigenesis, a number of cyto/chemokines has been found at detectable levels in ascites from eoc patients. among those molecules, il is the cytokine that has been most frequently associated with eoc carcinogenesis and progression. preclinical evidence has shown that il enhances tumor cell survival and increases resistance to chemotherapy via jak/stat signaling in tumor cells and il receptor alpha trans-signaling on tumor endothelial cells. in addition, il has pro-angiogenic properties, regulating immune cell infiltration, a stromal reaction, and the tumor-promoting actions of th lymphocytes. in patients with advanced disease, high plasma levels of il correlate with poor prognosis and elevated levels are also present in malignant ascites. treatment of eoc cells with the anti-il antibody siltuximab has been shown to reduce constitutive cyto/chemokine production and inhibit il signaling, tumor growth, the tumor-associated macrophage infiltrate, and angiogenesis in il6–producing intraperitoneal ovarian cancer xenografts. il stimulates inflammatory cytokine production, tumor angiogenesis and the tumor macrophage infiltrate in ovarian cancer and these actions can also be inhibited by a neutralizing anti-il ab in clinical studies. however, further knowledge on il6-expressing eocs is needed to select patients who are possibly responsive to il6-dependent therapies. we have recently found that il can be co-expressed together with plasminogen activator inhibitor - encoded by serpine in a subset of advanced stage serous eocs due to the activation of the ligand-dependent egfr/nfkb signaling cascade. ex vivo, using eocs from advanced-stage patients with malignant ascites at surgery, we observed co-expression of egfr, il and pai- in 57% of primary tumors and concomitant expression of both il and pai- in the corresponding ascites. computational analysis on four publicly available data sets of eoc gene expression showed a correlation between the expression of the il and serpine genes in advanced stage eoc patients, which in one case was associated with shorter progression-free survival. these results further highlight the involvement of il in the progression of eoc. herein, to give further insight in the biology of il6-expressing serous eoc we utilized a bioinformatics approach, described in the flowchart of figure  to identify il co-regulated genes and signaling pathway/s in which they are involved. first, we identified a list of genes representing a molecular signature for both advanced-stage and lmp serous eoc which recapitulate the so-called ‘early growth factor response’. we also identified an il6-correlated signature of seven genes involved in vascular thrombosis specific for advanced-stage serous eocs.  il expression significantly correlates with a defined gene set in advanced stage serous eocs pearson’s correlation analysis of seven data sets containing the expression profiles of samples from serous eocs was performed to identify genes whose expression was significantly correlated with il expression in each data set. correlation scores of each gene pair were computed using the r program essentially as described. for genes represented by multiple probes in the same array format, the probe with the highest correlation to il in the data set with the highest number of patients was chosen and considered for the other data sets when present. this analysis allowed the identification of genes whose expression positively correlated with il along the seven data sets with a p-value ≤ and a pearson’s correlation coefficient exceeding. a further analysis across the seven data sets yielded concordant correlated genes in at least four data sets. of note, of genes were correlated in data set i obtained with an affymetrix platform, and in data set vii obtained with an agilent platform. among the identified il6-correlated genes cxcl hbegf, serpine dusp zfp and ier were common to all data sets. the correlation between il and hbegf, an egfr ligand, and serpine encoding pai- is in agreement with our previously published results on co-expression of il and pai- in high grade eocs due to egfr activation. the majority of genes are associated to the biological process ‘proliferation’. among the genes associated with proliferation, there were a number of growth factor early response genes. the il6-correlated signature also included genes associated with ‘inflammation’, and the remaining genes were associated with ‘cell cycle and apoptosis’, ‘metabolism’ and ‘migration and invasion’. a biological functions were defined using genealacart tool. thus, we identified a gene signature of il correlated genes in serous eoc containing mainly proliferation-associated genes. advanced stage eoc-specific il6-correlated gene signature functionally associated with control of cell morphology and cardiovascular disease next, to determine whether the identified gene signature was specific for advanced stage eoc or could also be associated with lmp eoc, pearson’s correlation analysis to il was applied to gene expression data of lmp eocs reported in data sets i, ii, and iv. the density plot of il intensities showed a similar trend of expression in advanced stage and lmp eocs. the data obtained comparing advanced stage eocs and lmp eocs were similar in the three data sets, and were more reliable in data set i. among the above identified advanced-stage eocs il6-correlated genes, were also significantly correlated in lmp eocs, while genes were specific for advanced-stages only. among these genes, il has already been associated with aggressiveness and progression of malignant eoc, while the others have not previously been associated with eoc biology and clinical outcome. to provide insight into the possible biological significance of the 40-gene signature, functional analysis of positively correlated genes was carried out by ingenuity pathway analysis software. the top two functions, associated with the highest score network, were ‘cell death, cellular function and maintenance, hematological system development and function’ and ‘cell death, cellular development, cellular growth and proliferation’. when ipa analysis was performed on the seven-gene signature specific for advanced stage eocs, the top function, associated with the highest score network, was ‘cell morphology, cell function, cardiovascular disease’. as shown in figure  and listed in additional file all seven genes are included in this network together with genes already known to have a role in the progression of eocs such as vegf, the receptor tyrosine kinases egfr and her and the pi3k complex. in addition to the input genes, it is noteworthy that il is not present in the identified networks, but when added manually to each network establishes a connection with some of the correlated genes. the nfkb complex was included in networks n and n, highlighting its possible pivotal role in eoc progression. it thus appears that two signatures are related to il in eocs: a 33-gene signature common to advanced stage and lmp eocs and associated to control of cell growth and death, while the 7-gene signature, associated only to advanced-stage eocs likely presenting nfkb transcriptional activation, might be a determinant of tumor aggressiveness, and may be associated with a pathway regulating vascular thrombosis. an il6-correlated gene set recapitulates the early growth factor response to give further insight in the biology of eocs expressing il a gsea analysis was performed for each data set listed in table  by the “use a gene as phenotype” analysis, gsea first ranks the genes according to their correlation to il it then determines whether a priori defined set of genes, in this instance those belonging to the c curated catalogue of functional gene sets, are randomly distributed throughout the gene list or primarily found at the top or bottom. common significant gene sets obtained from gsea analysis of the two largest data sets were selected and analyzed in the other datasets. this yielded significantly enriched gene sets for all datasets. normalized enrichment scores and fdr values in the different datasets are listed in table  a literature search was conducted to identify signaling pathways previously implicated in the progression of eocs and/or in epithelial–derived malignancies. among the most significant gene sets, the bild_kras_oncogenic_signature which includes genes whose expression is induced by the activation of h-ras oncogene, was originally derived from the herein named data set iii and can be considered a positive control. three additional gene sets, amit_egf_response_60_hela, amit_egf_response_120_hela and nagashima_nrg1_signaling_up, were considered possible candidates of signaling pathways associated with eoc, and are associated with ‘growth factor response’. these gene sets comprise early response genes, i.e. the egr family members, and the negative feedback regulators of the growth factor signaling, i.e. zfp and klf the fifth selected gene set, named kim_wt1_target_up in some ways also recapitulates the growth factor response, since among wt target genes the egf family ligands ereg, areg and hbegf are included. nes normalized enrichment score, fdr false discovery rate. furthermore, among the wt target genes serpine was also identified in the same study. enrichment plots related to the above described gene-sets in data set i are shown in figure  it is of note that il is not included in the selected gene sets as well as other genes that are included in network identified by analysis using ipa. based on the results obtained by the above-described computational analysis and on our recent demonstration that il is up-modulated in eoc cells upon egf stimulation in time-dependent manner, in vitro validation of genes selected from the il6-correlated gene sets was performed with real time rt-pcr using total rna from egf-stimulated serous eoc cell lines. the il was up-modulated in all eoc cells analyzed upon egf stimulation. concordantly, 75%, 58%, and 75% of the gene transcripts were up-modulated in igrov oaw and skov cells, respectively. among the correlated genes common to data sets, cxcl hbegf, serpine and dusp were increased in all three eoc cell lines analyzed. additionally, nr4a a correlated gene in data sets, was up-modulated upon egf stimulation in all eoc cells. thbd and klf transcripts, associated with ’cardiovascular disease’ by ipa analysis, were up-modulated in of egf-stimulated eoc cells. in contrast, the mmp transcript, whose relevant protein is associated with invasion and tumor progression, was not up-modulated in egf-stimulated eoc cells. interestingly, in non-transformed ovary cells, named iose- htert, although il was slightly up-modulated by egf stimulation, only 25% of the transcripts analyzed were up-modulated. these data indicate that ligand-dependent egfr activation in serous eoc cells induces the transcription of genes correlated with il expression. discussion microarray technology has developed very rapidly, and it has become relatively easy to analyze the expression levels of thousands of genes within cancer cells. however, genes do not act in isolation, but each acts in complexes and builds networks and activated pathways that ultimately give rise to a specific cell phenotype. thus, the search of co-regulated genes applying bioinformatics approaches may spread light on the biology of a tumor and its development. previously, by applying this kind of ‘in silico’ approach on gene expression profiles of ovarian and thyroid carcinomas and melanomas, we have been able to identify novel signaling pathways activated in those tumors. the present study, by applying similar bioinformatics tools, highlights possible novel signaling pathways activated in il6-expressing eocs. among those, growth factor-dependent signaling was also experimentally validated in vitro in selected cellular eoc models. first of all, pearson’s correlation analysis allowed the identification of genes co-regulated with il in aggressive eoc providing evidence that co-regulated genes can encode proteins involved in common signaling pathways. to identify il6-coregulated genes we adopted thresholds which allowed to obtain a good balance among the statistical significance, the strength of the correlation and the biological reproducibility. furthermore, we performed the analysis on different data sets, containing the gene expression profiles of more than eoc samples, obtained on different array platforms, to increase the robustness on the bioinformatics results. we found a gene signature common to both advanced stage and lmp serous eocs, and another 7-gene signature specific for advanced stage eocs. the integration of the results obtained by ipa and gsea, allowed us to determine that all eocs, independently of their aggressiveness, co-regulate il together with genes associated with cell growth and early growth factor response, arguing for the hypothesis of common mechanism/s of transformation. only advanced-stage eocs appeared to be characterized by a scenario that involves genes such as fpr klf and thbd, to date associated with thrombosis and cardiovascular disease, thus suggesting that this pathway contributes to the growth and/or the spread of this type of tumor. our data indicate the existence of a biological interaction between il expression and that of the co-regulated genes as resulted upon ipa and gsea analyses. on the other hand, since knockdown of il- by specific sirna did not affect the amount of the transcripts analyzed, the regulation of the expression of the identified genes appeared not directly dependent to that of il although the il gene was not associated with the networks identified by ipa or with the gene sets selected by gsea, these results are in agreement with our previous observations in a subset of advanced stage eoc where ligand-dependent egfr activation induced nfkb-dependent transcription of il together with pai- encoded by the serpine gene. nfkb also emerged to be a possible transcriptional regulator of out of genes according to the reported informations, data which might further indicate that a growth factor-dependent nfkb signaling is activated in a subset of eoc. it is noteworthy that il and of the correlated genes were found up-modulated upon hr serum stimulation of quiescent keratinocytes. we can therefore argue that the activation of growth factor activated signaling can either directly or indirectly induce the expression of il and genes which likely play a role in the growth of eocs. furthermore, this growth factor-induced signaling pathway induces positive regulators of cellular function that are in turn regulated by negative feedback regulators such as zfp and klf. the hbegf gene, encoding for an egfr ligand, was also highly significantly correlated in all seven data sets analyzed, indicating the prevalence of ligand-dependent egfr activation. the regulation of growth factor signaling pathways by negative feedback is a universal mechanism for limiting the duration and intensity of signaling output. while negative feedback is a key component of normal cellular signaling, its role in cancer cells is more complex. indeed, the loss of some negative feedback regulators might contribute to tumor progression, but might also be expressed at considerably higher levels in oncogene-mutant tumors as observed in braf-mutated melanomas. interestingly, the presence of a feedback negative mechanism has also been associated with greater efficacy of growth factor receptor-targeted therapy. the fact that in eoc cells, with active egfr/nfkb/il signaling, egfr-targeted therapy was more effective might be due to the up-regulation of feedback negative regulators of growth factor signaling. taken together, these data suggest that the il6-associated signature might have a translational impact helping to select eoc patients who are likely responsive to egfr-targeted therapy. experiments are now ongoing to verify this hypothesis. nuclear expression of the wilm’s tumor suppressor is found in ose cells and in the majority of serous eocs. however, the corresponding gene, named wt has been never associated with il gene expression. wt is required for kidney development, and the report in which the relevant gene set has been derived particularly emphasized the finding that the genes encoding the egf family ligands ereg, areg, and hbegf may be transcriptionally regulated by wt orchestrating a fine-tuning of the egf signaling pathway. altogether these observations support that the egf signaling pathway is pivotal in the biology of eoc. the gene signature common to advanced stage serous and lmp eocs is not unexpected if one considers the theory that lmp eocs derive from serous low grade eoc with a borderline morphologic phenotype. however, if this is the case, advanced-stage and lmp tumors might share common genetic alterations that induce aberrant growth. in addition, in vitro validation experiments performed on gene transcripts of non-transformed surface ovary cells argue for the notion that the signature associated with a growth factor response is not expressed and/or egf-dependent in normal ovary cells. the bioinformatics approach also produced hypothesis-generating results. the association of the 7-gene signature with advance stage eocs is novel. at present only angiogenesis-related genes and proteins, such as vegf and its receptor, have a well documented role in eoc biology and are already well-exploited targets in the therapy of more aggressive eocs. our findings open new questions on the role of genes associated with thrombosis and cardiovascular disease in the progression of eocs. it has been recently hypothesized that low-dose aspirin as antithrombotic therapy may inhibit progression rather than the induction of eoc. indeed, aspirin and selective cox inhibitors could reduce progression not only by inhibiting prostaglandin production, thus reducing inflammation, but also by negatively modulating thrombosis-associated genes. therefore, the inhibition of both pathways synergistically might be an interesting approach to block the growth and dissemination of advanced stage eocs. mmp a gene of the 7-gene signature specific for malignant eocs and encoding the metallo-protease was present in network of ipa analysis, but was not in any of the gene sets selected by gsea analysis. mmps are key molecules of tumor cell invasion, including eocs and, since the majority of samples were advanced stage eocs, mmp could be a new player in the dissemination of these tumors and experiments are now ongoing to test its presence and role in advanced stage eocs.  by applying a bioinformatics approach we identified genes co-regulated with il expression in clinically-relevant subtypes of eoc, their interactions in networks and pathways as well as their functional association to growth factor response. il gene expression together with that of the correlated gene signature could help identifying eoc patient’s subgroups in which the identified signaling pathways might be biologically relevant during the progression of the disease and, in the long term, might represent new pharmacological targets. 
in agreement with these results, selected genes from the identified signatures were validated in vitro by real time rt-pcr in serous eoc cell lines upon stimulation with egf. the results obtained by gene set enrichment and ingenuity pathway analyses enabled the identification, respectively, of gene sets associated with ‘early growth factor response’ for the 40-gene signature, and a biological network related to ‘thrombosis and cardiovascular disease’ for the 7-gene signature. by pearson's correlation analysis we identified, in eocs, an il6-correlated gene signature containing genes mainly associated with proliferation. among the 40-gene signature egfr ligand hbegf, genes of the egr family members and genes encoding for negative feedback regulators of growth factor signaling were included. we applied bioinformatics tools on publicly available data sets containing the gene expression profiles of eoc samples. serous eocs, independently of their aggressiveness, co-regulate il expression together with that of genes associated to growth factor signaling, arguing for the hypothesis that common mechanism/s driven by egfr ligands characterize both advanced-stage and lmp eocs. only advanced-stage eocs appeared to be characterized by a scenario that involves genes which are so far associated with thrombosis and cardiovascular disease, thus suggesting that this pathway is implicated in the growth and/or spread of more aggressive tumors. il is a growth-promoting and anti-apoptotic factor, and high plasma levels of il in advanced stage eocs correlate with poor prognosis. il is the cytokine that has been found most frequently associated with carcinogenesis and progression of serous eocs. epithelial ovarian cancer is one of the most lethal gynecological cancers; the majority of eoc is the serous histotype and diagnosed at advanced stage. the objective of the present study was to identify il co-regulated genes and gene network/s in eocs. of genes were also significantly correlated in low malignant potential eocs, while genes, named c5ar fpr g0s il klf mmp and thbd were il6-correlated only in advanced stage eocs.
 epithelial ovarian cancer is one of the most lethal gynecological cancers; the majority of eoc is the serous histotype and diagnosed at advanced stage. il is the cytokine that has been found most frequently associated with carcinogenesis and progression of serous eocs. il is a growth-promoting and anti-apoptotic factor, and high plasma levels of il in advanced stage eocs correlate with poor prognosis. the objective of the present study was to identify il co-regulated genes and gene network/s in eocs.  we applied bioinformatics tools on publicly available data sets containing the gene expression profiles of eoc samples. by pearson's correlation analysis we identified, in eocs, an il6-correlated gene signature containing genes mainly associated with proliferation. of genes were also significantly correlated in low malignant potential eocs, while genes, named c5ar fpr g0s il klf mmp and thbd were il6-correlated only in advanced stage eocs. among the 40-gene signature egfr ligand hbegf, genes of the egr family members and genes encoding for negative feedback regulators of growth factor signaling were included. the results obtained by gene set enrichment and ingenuity pathway analyses enabled the identification, respectively, of gene sets associated with ‘early growth factor response’ for the 40-gene signature, and a biological network related to ‘thrombosis and cardiovascular disease’ for the 7-gene signature. in agreement with these results, selected genes from the identified signatures were validated in vitro by real time rt-pcr in serous eoc cell lines upon stimulation with egf.  serous eocs, independently of their aggressiveness, co-regulate il expression together with that of genes associated to growth factor signaling, arguing for the hypothesis that common mechanism/s driven by egfr ligands characterize both advanced-stage and lmp eocs. only advanced-stage eocs appeared to be characterized by a scenario that involves genes which are so far associated with thrombosis and cardiovascular disease, thus suggesting that this pathway is implicated in the growth and/or spread of more aggressive tumors. we have discovered novel activated signaling pathways that drive the expression of il and of co-regulated genes and are possibly involved in the pathobiology of eocs. epithelial ovarian canceril6microarraysbioinformaticsgrowth factor 