Background
Approximately 70% of breast cancer patients who have oestrogen receptor (ER) alpha-positive tumours receive adjuvant oestrogen deprivation therapy. Five years of aromatase inhibitor therapy produces a 40% reduction in 10-year mortality [
1]. However, while the annual risk of mortality for ER-negative breast cancer decreases following the first 5 years after diagnosis, the annual rate remains constant for ER
+ patients [
2]. In fact, women with ER
+ early-stage disease treated with 5 years of adjuvant endocrine therapy have a persistent risk of recurrence and death from breast cancer for at least 20 years after diagnosis [
3]. Molecular studies have demonstrated that nodal and distant metastases are highly similar to their matched primary tumours, implicating a continuation of the original cancer [
4‐
6]. However, the time between treatment and recurrence is often greater than that which can be explained by normal cell-doubling rates [
7], implying cancer cells remain dormant in the body before re-awakening.
Residual dormant cancer cells are hypothesised to persist either by withdrawing from the cell cycle and transitioning to a quiescence state or by continuing to proliferate at a reduced rate, counter-balanced by cell death [
8]. Reawakened dormant cells may become detectable after reaching a detection threshold or reactivated via increased angiogenesis, and/or escape from the inhibitory microenvironment or immune effects [
9,
10]. Dormancy is therefore considered a major mechanism underlying resistance to therapy, where dormant cells survive despite anti-proliferative oestrogen deprivation therapy.
Resistance to oestrogen deprivation therapy may occur at disease inception (de novo or innate resistance), but a larger proportion of patients acquire resistance during treatment (acquired/secondary resistance) [
11]. Several mechanisms of resistance to oestrogen deprivation therapy have been described previously [
12,
13]. However, the majority of these findings are based on preclinical data obtained from cell lines and animal models. It is therefore difficult to know if these accurately reflect molecular changes in patient tumours.
Expression profiling of clinical samples, measuring the effect of, or predicting response to, treatment has recently become feasible. However, experimental design issues, such as the difficulty in obtaining paired samples for comparison, particularly for longer time intervals, makes it difficult to study changes within tumours [
14]. For example, a previous study investigating tamoxifen failure compared samples from patients requiring salvage surgery with pre-treatment samples from an unrelated group of disease-free patients [
15]. More recently, sequential patient-matched samples have been successfully utilised to determine treatment-induced dynamic changes in tumours at 2 weeks to 3 months, demonstrating the effectiveness of this approach [
16‐
18].
For a variety of reasons, including being unfit for surgery, a proportion of patients receiving pre-surgical oestrogen deprivation therapy do not have their tumours excised following 3–4 months of treatment. These long-term endocrine-treated tumours represent a unique group that can inform how tumours respond to extended oestrogen deprivation in situ. Having initially shrunk in size, some tumours remain at a steady volume and appear dormant, whilst others subsequently begin to regrow. We have utilised this unique cohort of sequential samples from patients receiving extended neoadjuvant oestrogen deprivation therapy to characterise luminal breast cancer dormancy and acquired resistance as a novel clinical model.
Discussion
Understanding the mechanisms underlying the maintenance of and escape from dormancy have great importance considering that most cancer-related deaths are caused by metastasis rather than the primary tumour. In this study, we describe the first sequential patient-matched clinical dataset of extended oestrogen deprivation therapy in breast cancer. The results highlight the difficulty of distinguishing dormant and resistant tumours, with dynamic molecular changes of treatment being highly similar between the groups. However, comparative analysis revealed a set of genes significantly upregulated in resistant tumours compared with dormant tumours within the first months of letrozole treatment suggesting a predictive role for changes in DNA methylation.
Failure to reduce proliferation after 2 weeks of oestrogen deprivation therapy [
16,
35] may well identify patients that are innately resistant; however, acquired resistance remains a greater challenge in terms of identifying biomarkers and appropriate alternative or combination therapies [
36]. Many of the transcriptomic changes identified in long-term treated dormant tumours are shared by some, but not all, resistant tumours, providing further evidence of resistance heterogeneity [
37] where dormant tumours share similar molecular changes, but there are likely to be a variety of escape mechanisms that lead to acquired resistance.
In the present study, paired differential expression analysis demonstrated that dormant tumours continue to change under long-term treatment. Some of the identified dormancy-related pathways such as cell cycle arrest and senescence have established roles in metastasis dormancy [
38], further supporting the relevance of our clinical model, with the senescence-associated secretory phenotype (SASP) recently suggested to regulate breast cancer dormancy and relapse [
39]. As in short-term responsive tumours [
16], ECM organization and degradation were significantly upregulated in dormant tumours. ECM remodelling and its degradation by matrix metalloproteases (MMP) have previously been suggested to regulate the switch between dormancy and metastatic growth [
40]. Despite histological confirmation that each tumour sample contained at least 60% tumour, we acknowledge that the results presented are of intact whole tissue and potentially limited by minor variations in tumour cellularity or the proportion of stoma which could affect gene expression.
The most transcriptionally upregulated gene in resistant tumours
S100P, previously shown to be an inducer of breast cancer metastasis correlated with decreased survival [
41]. S100P, a small calcium-binding protein mediating Ca
2+-dependent signalling pathways, has distinct functions in normal tissue and cancer, including human embryonic development and breast cancer initiation [
42]. Recently,
S100P hypomethylation in blood was demonstrated to be inversely correlated with tissue
S100P expression and significantly associated with breast cancer, implicating
S100P as a potential diagnostic marker [
43]. High plasma
S100P levels have also been correlated with poor prognosis in metastatic breast cancer patients, with levels decreasing following treatment, suggesting a role of S100P in dynamic monitoring of response [
44]. In the present study, S100P gene expression and protein levels were significantly higher in resistant tumours after long-term treatment, as well as being differentially expressed before treatment, supporting its potential role as a therapeutic target [
45] and a predictive marker.
Comparative analysis of dormant and resistant samples after extended treatment revealed enrichment for a set of genes with a role in DNA methylation and histone acetylation/deacetylation. Epigenetic alterations are recognized to occur in breast cancer. DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors have been shown to exert encouraging effects on the disease [
46]. Recently, the potential role of epigenetic changes in regulating dormancy and reactivation state has been suggested to explain the reversible (on/off) nature of dormancy [
47].
Breast cancer “CpG island methylator phenotype” (CIMP), as revealed by genome-wide methylation analysis of metastatic breast cancers where a large number of genes are hypermethylated, has been suggested to be informative for metastatic potential [
48]. A significant correlation between pre-treatment global DNA methylation with neoadjuvant chemotherapy response in rectal cancer has been reported [
49]. Although DNA hypomethylation was the first epigenetic alteration identified in cancer, its molecular process and effects are not yet well understood [
50]. In addition, 5-hmC levels were shown to correlate with differentiation status, with higher levels when more differentiated [
29]. In addition, alterations in DNA methylation in LTED MCF7 cells have been previously reported [
51]. Our results provide evidence for the loss of a global DNA methylation process in resistant tumours and strengthen the case to use these models for further study. The global decrease in 5-mC may account for the observed reduction in 5-hmC levels since 5-mC is converted to 5-hmC. On the other hand, at the early-on time point, 5-hmC levels were significantly reduced with no significant change in 5-mC levels, suggesting an independent role of the 5-hmC mark. Hypomethylated cancer cells have been suggested to be selected to form tumours with increased malignancy [
50]. We suggest that hypomethylation in resistant tumours may reflect a de-differentiation process inducing stem cell-like cell formation. Determining the time point at which that hypomethylation starts, which would allow intervention before it starts to prevent resistance to therapy, needs further investigation.
The main genes significantly enriched for epigenetics-associated pathways in the present study are core histone (H3, H4, H2B) genes. Well-known epigenetics-associated genes such as DNMT were not differentially expressed in the present study. Therefore, it might be suggested that observed changes in histone gene levels may simply reflect the high proliferation rate in resistant tumours since transcription of these histone genes are replication-dependent and their mRNA levels increase during DNA replication [
52]. However, deregulation of histone H2A and H2B was associated with anthracycline resistance in breast cancer cells and reversed by HDAC small molecule inhibitors [
53]. Furthermore, upregulation of replication-dependent core histone proteins has been suggested to be a selective indicator of ER-mediated MCF7 cell proliferation regardless of the proliferation rate [
54]. Also, observed global loss of DNA methylation in resistant tumours suggests dynamic regulation of gene transcription under letrozole therapy. Therefore, histone upregulation and alterations in epigenetic pathways observed in our study may play a role in resistance to endocrine deprivation therapy, rather than simply mirroring the degree of proliferation.
Our results indicate alterations both in DNA methylation and histone modifications. HDAC inhibitors, which have been shown to regulate DNA methylation [
55], may be successful clinically as second-line drugs alone or in combination following oestrogen deprivation therapy failure as there is growing evidence for their tumour selective action [
56,
57]. A time-dependent role for HDACs in leukaemia has been shown [
58] and may also be critical in determining when to start HDAC inhibition therapy to successfully treat tumours resistant to oestrogen deprivation therapy. Whether or not the epigenetic alterations are triggers of re-awakening and if the timely use of epigenetic drugs can prevent acquired resistance warrants further investigation.