Background
Estrogen and progesterone signaling via their receptors play important roles not only in normal mammary gland development but also in breast cancer progression [
1,
2]. Gene expression profiling has revealed at least five subtypes of breast cancer: luminal A (LumA), luminal B (LumB), HER2, basal and normal [
3,
4]. Importantly, the immunophenotypic evaluation, i.e.
, the analysis of markers by immunohistochemistry such as estrogen receptor (ER), PR, Ki67, HER2 and basal cytokeratins (CK 5/6, CK14) is a useful surrogate of the gene expression defined-subtypes in the clinical setting [
5].
PR is expressed from a single gene as two isoforms, PRA and PRB. The two isoforms are expressed at similar levels in the breast, but the ratio can be altered in human breast tumors, with the PRA isoform predominating [
6]. Moreover, high PRA/PRB ratios predicted shorter disease-free survival in patients who received local therapy followed by adjuvant tamoxifen, indicating resistance to tamoxifen and underscoring the prognostic value of discriminating PR isoforms [
7,
8]. Importantly, preclinical studies with murine and human tumors and ex vivo human breast cancer tissue culture assays showed that antiprogestin responsiveness in breast cancer is determined by the PRA/PRB expression ratio, specifically, an inhibitory effect of the antiprogestin mifepristone is only obtained in tumors with higher levels of PRA than PRB [
9,
10].
In vitro, inducing a high PRA/PRB ratio in the T47D cell line conferred responsiveness to progestins to a set of genes involved in cellular metabolism and regulation of cell shape and adhesion. In accordance, progestin treatment resulted in reduced cell adhesion, which was significantly decreased even further when PRA was predominant [
11]. Other studies aiming to determine the relative contributions of PR isoforms functions of PRA and PRB have employed cell lines engineered to express only a single PR isoform [
12]. Due to common features, the mouse mammary gland is a useful model for normal human breast, and breast cancer [
1] and transgenic mice allow exploring hormone receptor actions in the gland. Transgenic mice carrying either an additional A form of PR (PRA transgenics) or the B form of PR show abnormal mammary gland features [
13]. In particular, mammary glands of PRA transgenics exhibit extensive lateral branching, ductal hyperplasia, a disorganized basement membrane and loss of cell-cell adhesion [
13‐
15]. Studies using the molecular markers for transformation, as defined by Medina [
16], revealed that these mammary glands contained at least two distinct populations of transformed epithelial cells. The ducts with normal histology contained cells resembling immortalized cells, while hyperplasias consisted of cells in later stages of transformation associated with early pre-neoplasias and exhibited increased epithelial cell proliferation [
15]. Similarly, loss of coordinate expression of PRA and PRB occurs early in human breast cancer progression [
17]. Therefore, evidence supports that misregulation of the PRA/PRB expression ratio can have major implications for mammary carcinogenesis.
In the present study, using the well characterized PRA transgenic mouse model, we sought to determine the full repertoire of target transcripts and pathways underlying the aberrant phenotype of mammary glands in PRA transgenics as compared with wild-type litter-mates in a relevant in vivo microenvironment under physiological hormone conditions. Further, using publicly available gene expression data sets of human breast cancer, we have explored the potential overlapping relevant pathways with breast cancer and identified novel putative biomarkers. Understanding the molecular context of deregulated PR action in the mammary gland may well accelerate the formulation of useful molecular descriptors for diagnosis, prognosis, and therapy of breast cancer.
Discussion
Changes in the native ratio of A to B isoforms of PR have major implications to normal mammary gland biology and also tumorigenesis. For this reason, several groups have previously used expression profiling to identify genes associated with PRA:PRB imbalanced ratio in breast cancer cell lines [
11,
26,
32,
33]. However, none have explored the transcriptional changes of preneoplastic lesions that are associated with PRA:PRB imbalance and tumor progression in an in vivo model. In this study, we took advantage of PRA transgenics, a previously described transgenic mouse model where PRA isoform is predominant. Using oligonucleotide microarray technology, we identified the complete repertoire of genes that are altered in expression in the abnormal mammary glands of PRA transgenics and characterized the associated pathways. Importantly, several of the DEG identified in this study (Table
1) had been previously reported as PR targets [
26].
Based on the distinct transcriptomics of PRA transgenics, we first identified by GO analysis an enrichment in the biological processes anatomical structure development and cell adhesion for upregulated and downregulated genes, respectively (Fig.
2a and
b). This is in accordance with the key morphological features of PRA transgenic mammary glands, including extensive lateral branching, the disruption in the organization of the basement membrane and a decrease in cell–cell adhesion [
13]. The influence of PRA:PRB ratio in cell adhesion has also been described in PR-positive T-47D breast cancer cells in which PRA can be induced to result in PRA predominance. Cell adhesion of T-47D cells was decreased upon progestin treatment and reduced even further with PRA predominance [
11]. Another enriched biological process identified in this study for downregulated genes was transmembrane transport, and the cell membrane itself was enriched amongst the cellular compartments. Similarly, Richer et al. found an extensive number of genes involved in membrane-initiated events that were regulated by PR isoforms in response to progesterone, thus stressing the membrane as an important target of progesterone action [
33]. Also, more recently, SLC- mediated transmembrane transport was found amongst enriched pathways in human breast PRA high tumors as compared to PRB high tumors [
10].
Then, we used GSEA to identify pathways and unifying themes. The GSEA method focuses on gene sets rather than a handful of high scoring genes at the top and bottom (which can suffer from arbitrary cutoff regarding fold-change or significance) giving more reproducible and easy to interpret results [
20]. When necessary, we plotted enrichment maps of gene sets to aid interpretation [
21]. Interestingly, we found a positive correlation of PRA transgenics with gene sets comprising amplicons previously identified in human breast cancer (Fig.
3a). One example is amplicon 16p13, which was previously correlated with luminal breast cancer subtype and comprises effector proteins such as proteases [
28]. For genes downregulated in PRA transgenics, we identified three clusters based on the enrichment map (Fig.
3b). Not surprisingly, one cluster (ii) included gene sets related to mammary gland morphological changes or breast cancer subtypes. For example, a set comprising genes downregulated in ductal carcinoma vs normal ductal breast cells identified by laser microdissection and microarray analysis [
34] and another set of genes that were downregulated in HMLE cells (immortalized nontransformed mammary epithelial) cells after loss of function of E-cadherin (CDH1) achieved by RNAi knockdown or by expression of a dominant-negative form [
35]. Of note, mammary glands of PRA transgenics exhibited diminished E-cadherin in a disorganized pattern [
13]. Another region in the map (ii) clustered gene sets related to estradiol or tamoxifen response like one set of genes downregulated in breast cancer SUM44/LCCTam cells resistant to 4-hydroxytamoxifen relative to the parental sensitive cells [
36]. Importantly, in PR-positive breast cancer patients who received local therapy followed by adjuvant tamoxifen, high PRA:PRB ratios predicted shorter disease-free survival, indicating resistance to tamoxifen [
7]. Our third (iii) identified cluster included gene sets comprising targets of polycomb complexes. In particular, Polycomb Repression Complex 2 (PRC) targets that possess H3K27me3 mark in their promoters and are bound by SUZ12 and EED Polycomb proteins [
37]. This suggests that PRC2 may be an upstream regulator accounting for the high number of downregulated genes in PRA transgenics. Interestingly, H3K27me3 mark has been positively associated with the LumA subtype compared to all other subtypes in a cohort of breast cancer patients [
38].
We then performed GSEA analysis using the Hallmark collection (Table
2). We found positive enrichments mainly in metabolic pathways for the PRA transgenics phenotype. Changes in metabolism during tumorigenesis are well known. Our analysis suggests metabolic plasticity, as oxidative phosphorylation in addition to glycolysis were significantly enriched, and this adaptability may be important as tumorigenesis progresses [
39]. Pathways negatively correlating with PRA transgenics phenotype pointed to an anti-inflammatory effect. PR action has been linked previously with inhibition of inflammatory response in breast cancer cells [
40] and myometrial cells [
41]. However, the uterine phenotype of PRA transgenics included endometritis and pelvic inflammatory disease (together with hyperplasia) [
42], therefore effects on inflammation may be context-dependent in PRA transgenics. Recently, Cai et al. analyzed gene expression from four distinct stages of mammary tumor progression using the MMTV-PyMT mouse model and found similar enriched hallmark pathways amongst up and downregulated genes in the hyperplasia stage [
43]. For example, apical junction, epithelial to mesenchymal transition, UV response, KRAS signaling up, IL2 STAT5 signaling and hypoxia (all also enriched in the present study) were enriched in the down-regulated DEGs at normal to premalignant (hyperplasia) transition in the MMTV-PyMT model. Importantly, most DEGs identified in the same study in the late carcinoma stage first appeared in the much earlier hyperplasia stage, consistently with previous human cancer studies [
29,
30]. This prompted us to compare the hallmark pathways identified for PRA transgenics to those obtained using the same methodology in a much larger data set of human breast cancer samples [
22,
23]. Interestingly, we found significant overlapping between pathways exclusively with luminal breast cancer subtypes (Table
4). Rojas et al. have recently classified human breast tumors according to their PRA:PRB ratio (high versus low) by western blot detection and predicted according to the PAM50 gene set that PRB-High and PRA-High tumors were either luminal B or A phenotypes, respectively [
10]. It would be interesting to determine by the same method the intrinsic subtype based on PRA transgenics gene expression profile, as at least by our approach overlapping with luminal B subtype is predicted according to pathway analysis. Finally, we explored the potential prognostic value of common candidate genes from the most significantly enriched pathways and found an association with worse relapse free survival and high PDHB (upregulated in PRA transgenics, Fig.
4) or low LAMB3 (downregulated in PRA transgenics, Fig.
5) for luminal breast cancer subtypes. To the best of our knowledge, this is the first report of the potential prognostic value of this particular laminin subunit and PDHB in breast cancer.