Although often ignored and under-appreciated, breast cancer patients suffer from potentially debilitating stress, anxiety, depression, and impaired cognitive function [1
]. Stress has been demonstrated to result in an increased incidence of cancer recurrence [3
]. On the contrary, breast cancer patients with no stressful or traumatic life events have significantly longer disease-free intervals compared to patients who have experienced these events [3
Stress activates the hypothalamic–pituitary–adrenal axis with corticotropin-releasing factor produced in the hypothalamus, which stimulates the release of adrenocorticotrophic hormone (ACTH) from the anterior pituitary. ACTH signals the adrenal cortex to produce glucocorticoids—the stress hormone ‘cortisol’ secreted by the zona fasciculata of the adrenal glands. Cortisol levels are elevated in patients with breast cancer and the diurnal cortisol rhythm is a predictor of breast cancer survival [4
]. Cortisol generates physical response to stress by binding to its cytoplasmic receptor, glucocorticoid receptor (GR), which is the transcription factor encoded by the NR3C1
], thus promoting “stress response” [6
The role of GR activation has been reported to be different between breast cancer subtypes, namely, estrogen receptor (ER)-negative and ER-positive breast cancer. Activation of GR in ER-negative human breast cancer cell lines has been shown to promote cancer cell survival, chemotherapy resistance, and increased tumor growth in a pre-clinical xenograft model [8
]. A retrospective meta-analysis in primary breast tumors showed that high gene expression of GR (NR3C1
) in the bulk tumor was associated with significantly worse relapse-free survival (RFS) among ER-negative early stage breast cancer, but better survival in ER-positive breast cancer. It was speculated that this difference is due to an interaction of the GR and ER [10
The levels of stress hormones, cortisol and corticosterone, were higher in the plasma of mice with metastatic breast cancer than in healthy controls. Obradovic et al. showed that increase in stress hormones during breast cancer progression results in activation of GR at distant metastatic sites, increased colonization and reduced survival [11
]. Studies have shown that strategies to antagonize GR signaling can sensitize ovarian, prostate and triple negative breast cancer (TNBC) cell lines to chemotherapy. Therefore, inhibition of the GR pathway is being investigated in clinical trials combining GR/PR antagonist mifepristone with nab-paclitaxel [12
Although the role of GR activation in chemoresistance and enhanced aggressive phenotype have been studied both in vitro [13
] and in vivo [13
], both models lack immune cells. It is well known that TNBC have more immune cell infiltration than ER-positive tumors [14
]. Immune cells, including T-cells, B-cells, monocytes, neutrophils, and macrophages, also express the GR, in addition to cancer cells in the bulk tumor [15
]. In addition to the downstream effects of GR activation on tumor cells, over the last few years, there has been interest in understanding GR expression on immune cells in the bulk tumor [16
] and the impact of its activation [17
Recent computational biological analyses of transcriptomic data of bulk tumors allow us to investigate human tumor immune microenvironment (TME) in large cohorts. Here, we hypothesized that not only cancer cells, but immune cells in the TME also contribute to the GR expression of the bulk tumor, which may contribute to differences in outcome.
In this study, we examined the tumor microenvironment differences between GR-high and GR-low breast cancer and explored role of immune cells in GR expression of the bulk tumor to explain the disparities in outcomes in the two cohorts. This is in addition to the well-known direct transcriptional role of GR on tumor cells which plays a critical role in clinical outcomes.
Our study shows that GR-high breast cancer has better survival compared to GR-low breast cancer, particularly in ER-positive/HER2-negative breast cancer. This is consistent with previous retrospective studies showing that GR expression in ER-positive breast cancer is associated with better outcomes [10
]. On the contrary, previous studies have shown that GR expression in ER-negative breast cancer was associated with worse RFS. Our analysis showed that high GR expression was associated with better outcomes in TNBC in METABRIC but not validated in TCGA where only a trend towards improved survival was seen with no statistical significance. Our findings in TNBC subtype did not replicate results from previous publications probably due to the different data cohort (TCGA and METABRIC) and methodology for ER-positivity used here [10
]. As published by Conzen et al. in Cancer Research
, 8 Gene Expression Omnibus (GEO) studies were combined and expression of ESR1 was used to categorize samples into specific subtypes with 1378 patients (1024 ESR1 positive and 354 ESR1 negative) [10
]. A combination of studies is less ideal than using a single cohort that has been curated and standardized in uniform manner, because of different sequencing techniques used (variable standardization). In addition to these differences, we investigated the contribution of immune cells to explain this discrepancy in outcomes.
It is well known in literature that the presence of T-regs is associated with poor RFS and worse outcomes in breast cancer [21
]. To our knowledge, this is the first study to find significant differences in the immune cell landscape in the GR-high vs. GR-low breast cancer. In our current study, we observed that there are a lesser number of immunosuppressive T-regs in GR-high tumors. On the other hand, we also found lower anti-tumor T-follicular helper cells [22
] in the GR high tumors. Although there are both lower immunosuppressive and anti-tumor cells, overall we observed a higher cytolytic activity in GR-high tumors. It is interesting to note that although we observed enrichment of TGFβ signaling in GR-high breast cancer, there were lower Tregs in this group. Prior studies have shown that in addition to TGFβ, other cytokines such as IL-10, IL-4 and IL-13 are also involved in Treg generation and induction, and it is possible that these other cytokines could be implicated in regulating Tregs in our study [23
]. In addition, we found that GR-high breast cancers have a higher immune response with higher IL-2 pathway and apoptosis and lower proliferation, explaining improved survival. However, this is in addition to higher TGF-β score, KRAS and EMT pathways. TGF-β may promote or inhibit tumor progression [24
], whereas KRAS and EMT pathways are associated with worse prognosis, though there is still a lack of data for the role of KRAS in breast cancer [26
]. It is interesting to note that while KRAS and EMT pathways that result in aggressive tumor biology are enriched in the GR-high group, immune pathways IL2 and apoptosis are also enriched, which may play a role in influencing overall survival. The importance of anti-tumor immune response in influencing overall prognosis and outcomes in breast cancer is evident from literature [28
]. Prior studies have also shown a similar GR-associated modulation of immune response pathway genes [10
] in ER-negative subtype, but we observed a difference in TME both in ER+/HER2 negative and TNBC subtypes. As is published in the literature, in addition to a direct transcriptional role of GR on the tumor cells, which could be mediated by EMT pathways elevated in the GR-high group, the contribution of immune cells to outcomes should also be acknowledged because of a potential interaction of GR activity with the TME.
Breast cancer patients suffering from stress have shorter survival compared to patients who do not report stress [3
]. Stress mediated cortisol release in peripheral blood acts on the cytoplasmic GR resulting in cancer cell survival, chemotherapy resistance, and increased tumor growth in pre-clinical models as well as plays a role in regulation of immune system [8
]. Studies have investigated the variation in the expression of GR on immune cells [15
] with stress and have shown different findings. Even among the immune cells, the relative expression of GR varies among different cells, with higher expression in eosinophils, followed by granulocytes, T lymphocytes and NK cells (p
< 0.05) [31
], however, no correlation was observed between serum cortisol and GR expression on the leucocyte subpopulations [31
]. On the other hand, another study showed reduced peripheral expression of the GRα isoform on the peripheral blood cells in individuals with post-traumatic stress disorder: a cumulative effect of trauma burden [32
]. Although our study showed that CD8+
T cells have higher GR expression, however, we did not see any difference in their distribution between GR-high and GR-low breast cancer. Similarly, although macrophages have minimal GR expression, a moderate positive correlation of GR expression with macrophages was observed using TIMER, however, this correlation was not validated in more robust deconvolution algorithms. At the same time, we did not see any difference in macrophage distribution between GR-high and GR-low breast cancer. This leads us to hypothesize that immune cells in addition to macrophages and CD8+
T-cells are likely to be contributing to the GR signature of the bulk tumor, though none of the immune cells were independently elevated in GR-high breast cancer. It is well known that GR signaling influences functions of different immune cells. Glucocorticoids exert anti-inflammatory activity by inhibiting neutrophil rolling, adhesion and activation; they inhibit dendritic cells to activate T-cells; favor T-cell apoptosis by acting on T-helper 1 (Th1) cell by decreasing T-bet transcriptional activity and suppressing the production of pro-inflammatory molecules IL-2 and IFN-γ that favors T-reg expansion [33
Single-cell RNA sequencing provides a new platform to understand the dynamic ecosystem that comprises of tumor cells, fibroblasts and immune cells. Gene expression data from bulk tumors is indispensable and continues to dominate the clinical and translational settings, however, TCGA designs have been focused on the cancer cells (high amount of cancer cells were one of the criteria of the sampling of the tissue), whereas single-cell RNA sequencing data can capture the gene expressions of cells in the surrounding stroma such as immune cells [34
]. Single-cell sequencing technologies hold the potential to revolutionize the field of cancer [36
]. We pursued analysis of single cell sequencing data in order to analyze the contribution of GR expression on the immune cells (contributing to the bulk genomic and transcriptomic signature). Interestingly, our single-cell sequencing data show that immune cells express significantly higher GR compared to other tumor and stromal cells, and thus, contribute to the GR expression of the bulk tumor.
Since immune cells also express GR, we speculate that modulation of GR signaling in the presence of cortisol may cause their activation/suppression, which may additionally contribute to the different outcomes in GR-high versus GR-low groups. Our observation that there are lesser number of immunosuppressive pro-cancerous T-regs in GR-high tumors may consolidate the findings from a previous study [32
] as patients with less stress (less cortisol) may have higher GR expression with lower number of T-regs, thus higher CYT and hence, improved survival. This provides a hypothesis that in addition to a direct transcriptional role of GR on tumor cells and interaction with ER, there may be an additional role of immune cells in GR-high vs. GR-low tumors in influencing prognosis and potentially as a therapeutic strategy in addition to targeting the GR expression on tumor cells. Our hypothesis is further strengthened by the recent approval of checkpoint inhibitors in breast cancer with high PD-L1 expression on immune cells [37
], further highlighting that immune-mediated pathways are crucial and present an excellent opportunity for targeted approaches to overcome underlying immunosuppression in breast cancer and improve outcomes.
Our study limitations include analysis from a publicly available database and also limited data interpretation by lack of a mechanistic approach and causality association as this study does not contain in vitro and in vivo data. The finding of different immune cell subpopulations in GR-high and GR-low groups and the contribution to outcomes is hypothesis-generating and needs mechanistic validation. Future work needed to advance this field further should focus on investigating if GR expression correlates with GR signaling by analyzing downstream pathways in different subtypes of breast cancer, especially receptor-tyrosine-kinase-like orphan receptor 1 (ROR1) signaling, which has been shown to be associated with aggressive disease and decreased survival in breast cancer [11
]. In addition, this would also help elucidate if higher GR expression on immune cells corresponds to increased or decreased sensitivity towards GR signaling/activation and immunosuppression.