This is the first study to compare gene expression between primary EOC tumors and their matching omental metastases using RNA-seq, allowing more sensitive and deeper characterization of transcriptome compared to microarray [
9]. In line with previous array-based findings, we find that the gene expression profiles of metastases differ from those of the primary tumors [
6,
7]. In addition, our analysis confirms the metastasis signature being enriched for TP53 pathway and functions related to cell adhesion and proliferation [
6‐
8]. Of the differentially expressed genes in our study,
NR1H4, CADPS, STAR, SFRP2 and
EPYC were also observed to be differentially expressed in the similar direction in the earlier studies [
6,
8]. In contrast to previous studies, our analysis identified repression of embryonic developmental genes as the biggest group of genes repressed during metastasis formation in ovarian cancer. Indeed, many of the embryonic developmental genes were also found to be highly expressed in ovarian cancer compared to many other cancers in the TCGA data, including
FOXL2, GATA4, NR5A1, AMHR2, MAL and
WIPF3. Of these, the first three were further identified as potential upstream regulators that could explain the observed gene expression patterns. Accordingly, the
GATA4 has been shown regulate genes involved in embryogenesis and development of the female reproductive organs, testes, GI-tract, heart and lungs [
24]. Loss of this tumor suppressor gene expression has been connected to certain ovarian cancer subtypes in several studies: serous [
25], clear cell [
25,
26] and endometrioid [
25] ovarian cancers, while mucinous ovarian cancer expresses
GATA4 [
25]. This is in concordance with our finding that
GATA4 is downregulated in our metastatic gene signature in HGSOC. Statistically significant higher methylation leading to the loss of
GATA4 expression in endometrioid type compared to serous ovarian adenocarcinoma has been reported [
27]. However, no correlation between
GATA4 expression and patient age, histologic type, histologic grade, stage of the disease or survival in ovarian surface epithelial carcinomas has been reported [
28]. Another upstream regulator,
NR5A1 transcription factor, was also downregulated in omental samples. It encodes a human steroidogenic factor 1-protein (hSF1) that is involved in gonad development in both males and females [
29]. hSF1 expression has been found to be significantly lower in ovarian cancer than in normal ovarian tissue [
30] and mutations in
NR5A1 are associated with primary ovarian insufficiency [
31]. The third upstream regulator identified in our analysis was
FOXA2, that has demonstrated favorable prognosis based on TCGA data [
32] and was predicted to regulate six genes that were downregulated in omental samples vs primary tumor (
DLK1, GATA4, MAFA, MYOCD, NR1H4 and
WNT5). However,
FOXA2 was not differentially expressed in our data but rather another member of the FOX-family that encodes for transcription factor that is involved in all stages of ovarian development and function,
FOXL2 [
33]. Whether FOXL2 acts to regulate predicted FOXA2-targets in ovarian cells remains to be studied. Interestingly, C134W mutation in this gene is indicated to be connected to granulosa cell tumors [
34]. In a recent study
FOXL2-positive cells were found mainly in primary and secondary ovarian tumors and very few in peritoneal seeding sites suggesting that local tissue environment could be responsible for its omental downregulation [
35]. On the other hand, the changes in gene expression can also be due to changes in proportions of cell types as recently indicated by a decrease in cancer epithelial cells in ovarian cancer metastases [
36]. Future studies incorporating single cell technologies are needed to evaluate the potential of the identified factors as prognostic or therapeutic targets versus cell-subtype markers.
The identification of different ovarian cancer subgroups could allow for more personalized treatments and is therefore heavily investigated. Previous molecular subtyping systems defined by TCGA and Tothill studies [
10,
11] have demonstrated the existence of four molecular HGSOC subtypes represented earlier by [
10] and termed them ‘mesenchymal’ (C1), ‘immunoreactive’ (C2), ‘differentiated’ (C4) and ‘proliferative’ (C5) [
11]. Different molecular subgroups did not have prognostic significance in the TCGA study, but later on it was demonstrated that the proliferative and mesenchymal subtypes are associated with the poorest prognosis [
37] and mesenchymal subtype with the lowest optimal-debulking rates [
38]. In our study, the metastatic tumors had a gene expression signature more similar to the mesenchymal C1-group in the TCGA study compared to primary tumors. In line with this [
10], the differentially expressed genes in our metastasis samples were involved in processes related to extracellular matrix signalling and cell cycle, suggesting that regulation of connective tissue deposition is upregulated in metastases. Recent study has also demonstrated that this subtype demonstrates upregulation of the TGF-β pathway [
38]. Similarly, several other expression studies have reported that TGF-β pathway activities are associated with worse clinical outcomes and ovarian cancer metastasis [
31,
38‐
40]. Therefore, tumours with the mesenchymal gene expression pattern might be considered for future trials containing TGF-β inhibitors.
Finally, survival analysis based on gene set enrichment analysis of TCGA primary tumors expression profiles revealed that the differentially regulated genes identified in this study could be indicative of poorer survival. This is in line with previous report based on 19 matched primary and omental metastatic tumors from 3 different serous adenocarcinoma types [
8]. In contrast, another study showed that many good prognosis genes were more highly expressed and poor prognosis genes lower expressed in the peritoneal metastasis vs primary tumor, indicative of the metastatic lesions remaining closer to normal tissue [
7]. This is in line with the expression patters of
MAL and
FAM19A2 in our analysis. However, among the five other genes with prognostic value, genes associated with better prognosis were downregulated (
GATA4, AMHR2 and
PCSK6) and genes with poorer prognosis were upregulated (
PAX5 and
SFRP2) in the metastatic samples. This could reflect subtype differences of the EOCs, as patients in our study were limited to HGSOCs only. Recent reports have also identified markers related to recurrence in ovarian cancer primary tumors. These further identified networks related to TP53 and TGF-β signaling, cell cycle, leukocyte migration and cellular adhesion [
41,
42]. Evidently, deciphering the molecular mechanisms and similarities of metastatic transformation and recurrence of primary tumors will be important for understanding the pathogenesis of the disease and to improve the treatment, especially in advanced stage. Despite the exploratory nature of our study, limited by low sample amounts and overall small effect on survival, our study provides many candidates that warrant future research and replication in other independent cohorts. Overall, our analysis reveals novel aspects of metastatic transformation of HGSOC, with potentially important implications for prognosis and therapy.