key: cord-0899125-0vdk64se
authors: Annett, Stephanie; Fox, Orla Willis; Vareslija, Damir; Robson, Tracy
title: Dexamethasone promotes breast cancer stem cells in obese and not lean mice
date: 2022-03-14
journal: Pharmacol Res Perspect
DOI: 10.1002/prp2.923
sha: ffd342e7ab040b09a5b9f5d9bea326c7100d3b86
doc_id: 899125
cord_uid: 0vdk64se

Obesity is highly prevalent in breast cancer patients and is associated with increased recurrence and breast cancer‐specific mortality. Glucocorticoids (GC) are used as an adjuvant in cancer treatment and are associated with promoting breast cancer metastasis through activation of stemness‐related pathways. Therefore, we utilized the synergetic allograft E0771 breast cancer model to investigate if treatment with GCs had differential effects on promoting cancer stem cells in lean and diet‐induced obese mice. Indeed, both lean mice treated with dexamethasone and obese mice with no treatment had no effect on the ex vivo colony‐forming ability, mammosphere formation, or aldehyde dehydrogenase (ALDH) bright subpopulation. However, treatment of obese mice with dexamethasone resulted in a significant increase in ex vivo colony formation, mammosphere formation, ALDH bright subpopulation, and expression of pluripotency transcription factors. GC transcriptionally regulated genes were not altered in the dexamethasone‐treated groups compared to treatment controls. In summary, these results provide initial evidence that obesity presents a higher risk of GC‐induced cancer stemness via non‐genomic GC signaling which is of potential translational significance.

Steroids are routinely used in patients with solid tumors as adjuvant therapy to manage tumor and treatment-related symptoms.

However, a meta-analysis of >80 000 patients found that the use of steroids in solid tumors was associated with reduced overall and progression-free survival. 1 One explanation for this is the observation is that glucocorticoids (GCs) promote a stem cell phenotype. [2] [3] [4] In breast cancer, plasma levels of cortisol (an endogenous GC) increase during breast cancer progression, inducing activation of the glucocorticoid receptor (GR) resulting in increased metastatic colonization. 5 Worryingly, treatment with dexamethasone (a clinically used GC drug) also activated the GR causing increased metastatic burden and decreased survival. 5 Obesity affects more than half a billion adults worldwide and it is a well-known risk factor for breast cancer and an indicator of poorer prognosis. 6, 7 Indeed, obese breast cancer patients have an increased relative risk of recurrence of 40% to 50%, however, the underlying biology behind the link between obesity and breast cancer progression remains unclear. 8 Obesity causes chronic inflammation in the adipose tissue and elevated Abstract Obesity is highly prevalent in breast cancer patients and is associated with increased recurrence and breast cancer-specific mortality. Glucocorticoids (GC) are used as an adjuvant in cancer treatment and are associated with promoting breast cancer metastasis through activation of stemness-related pathways. Therefore, we utilized the synergetic allograft E0771 breast cancer model to investigate if treatment with GCs had differential effects on promoting cancer stem cells in lean and diet-induced obese mice. Indeed, both lean mice treated with dexamethasone and obese mice with no treatment had no effect on the ex vivo colony-forming ability, mammosphere formation, or aldehyde dehydrogenase (ALDH) bright subpopulation. However, treatment of obese mice with dexamethasone resulted in a significant increase in ex vivo colony formation, mammosphere formation, ALDH bright subpopulation, and expression of pluripotency transcription factors. GC transcriptionally regulated genes were not altered in the dexamethasone-treated groups compared to treatment controls.

In summary, these results provide initial evidence that obesity presents a higher risk of GC-induced cancer stemness via non-genomic GC signaling which is of potential translational significance.

breast cancer, cancer stem cells, glucocorticoids, obesity, tumour initiating cells levels of cortisol in both the local adipose tissue and the systemic circulation. 9, 10 Given the role of GCs in breast cancer metastasis, we hypothesized that obesity may increase the risk of GC-induced breast cancer reoccurrence by further promoting cancer stem cells. was determined as previously described. [11] [12] [13] RNA was isolated using the RNAeasy kit (Qiagen) and gene expression was quantified by real-time PCR with primers listed in Table S1 .

Female mice fed a high-fat diet (HFD) for 12 weeks were significantly heavier than mice fed a normal diet ( Figure 1B ; p < .0001).

Contrary to the previous reports of diet-induced obesitypromoting E0771 tumor growth 14, 15 we found no difference in the tumor growth rate between any of the groups; normal diet and no treatment (n = 8), normal diet and dexamethasone (n = 7), HFD, and no treatment (n = 6), HFD and dexamethasone (n = 5) ( Figure 1C ). GCs were previously reported to have antiangiogenic activity in solid tumors 16 and, therefore, we measured the gene expression of the endothelial marker Cd31 (also known as Pecam-1). There was no difference in Cd31 gene expression between mice on a normal diet treated with or without dexamethasone ( Figure 1D ). However, mice fed a HFD had a significantly increased expression ( Figure 1D to mice on a normal diet treated with dexamethasone. ALDH has been extensively studied in breast cancer as a marker for cancer stem cells. In addition, it is associated with chemoresistance and poor survival in patients. 17 Mice on a normal diet, or a HFD, did not have an increased ALDH + bright population (Figure 2A) . However, in line with previous results mice fed a HFD and treated with dexamethasone had an increased ALDH + bright subpopulation compared to the other treatment groups (Figure 2A ; p = .0302).

Next, we measured the pluripotency transcription factors Nanog, It has been reported that GCs increase stemness and metastasis through activation of the non-canonical Wnt signaling axis 4,5 and therefore we investigated if Ror1, and its ligand Wnt5a, was also activated in our tumors. Treatment with dexamethasone on a normal diet increased expression of Wnt5A ( Figure 2C ; p = .0506), but this was not increased by HFD ( Figure 2C ). Ror1 expression was not altered in any of the treatment groups ( Figure 2C ). In addition, genes classically regulated by GCs, Nrcl1, and Fkbp51, 18 were not induced in the tumors of any group ( Figure 2D ). Together, this indicates that dexamethasone promotes a cancer stem cell phenotype in obesity through upregulation of pluripotency transcription factor via non-classical GC signaling.

Adjuvant GC drugs (such as dexamethasone) are routinely used during the treatment of cancer to mitigate the undesirable effects of chemotherapy such as nausea and fatigue and to stimulate appetite. 19 In addition, they reduce hypersensitivity reactions to some chemotherapy agents and enhance tolerability to allow higher chemotherapy doses and more frequent cycles. 19 However, studies have revealed that GCs can promote cancer progression, nevertheless, the literature remains conflicting, and it is not clear how GCs either promote or inhibit tumor progression in different cancer types and by different mechanisms. Here, for the first time, we describe that obesity also has a profound effect on the ability of dexamethasone to promote the cancer stem cell phenotype, that does not occur in lean mice. Interestingly, overall, we found that obesity or dexamethasone alone had a limited effect on promoting cancer stem cells ( Figure 1F , Figure 2A ). This is in contrast to previous reports demonstrating that both obesity and dexamethasone alone promote metastasis via stem cell-related signaling. 3, 4, 14, [20] [21] [22] [23] In our study, we found that dexamethasone promoted expression of the pluripotency transcription factor Sox2 in both normal diet and obese mice ( Figure 2B) , potentially indicating that a longer treatment time may promote stemness in lean mice. On the contrary, several studies have shown that dexamethasone decreases Sox2 and thereby inhibits cancer stem cells. 24, 25 In addition, similar to our study, dexamethasone did not promote Ror1 expression in pancreatic cells 24

( Figure 2C ). Together these results indicate that GCs are a regulator of Sox2 expression, however, its activation or inhibition is dependent upon the cell type and/or microenvironment. Indeed, a recent preprint on BioRxiv demonstrates, through single-cell transcriptomics, that following acute GC exposure, cerebral organoids elicit differential effects depending on cell type. 26 Similar experiments utilizing single-cell sequencing will aid our understanding of the cell typespecific effects of GCs within the tumor microenvironment. Patients living with obesity make up a substantial proportion of individuals with breast cancer, however, they may not benefit equitably from established therapies. It is well-described that there is a reduced efficacy of cancer treatment among obese patients, particularly for chemotherapy in which the dose is often based on ideal body weight rather than actual body weight because of toxicity concerns. 27 We now provide initial evidence that GCs may have an altered mechanism of action in obesity resulting in expansion of breast cancer stem cells, which can promote recurrence and metastasis. Initially, we hypothesized that obesity-induced activation of classical GC signaling which may promote stemness. However, known GR transcription targets Nr3cl1 (glucocorticoid receptor) and Fkbp51 were not altered in the tumors following dexamethasone treatment ( Figure 2D ). This

indicates that dexamethasone use in obesity may promote stemness via a non-genomic signaling mechanism. This may involve either membrane or cytoplasmic GR that does not require nuclear translation and subsequent GR-mediated transcription. GR interacts with several kinases such as JNK, Src, PI3K, 19 and this may play an important role in the GC regulation of stemness, however, the nongenomic activities of GCs are not well understood. In summary, we provide pre-clinical evidence that administration of dexamethasone in obesity results in an increased cancer stem cell-like subpopulation in mice, and GCs may promote tumor reoccurrence in breast cancer patients also living with obesity.

I would like to thank all the technical staff that aided with the completion of this study during the COVID-19 pandemic.

No conflicts of interest.

The authors were involved in the following tasks: SA (conception and design of the study, data acquisition, data interpretation, statistical analysis of the manuscript, data interpretation, writing, and F I G U R E 2 High fat diet and dexamethasone promotes an ALDH + bright subpopulation and pluripotency transcription factors. (A, B) Ex vivo analysis of an ALDH bright subpopulation of cells in excised E0771 tumors from mice fed either a normal or high fat diet and/or treated with dexamethasone (0.1 mg/kg) in the drinking water for 5 out of 7 days for 3 weeks. Expression of mRNA (C) Sox2 and Nanog, (D) Ror1 and Wnt5a and (E) Nr3cl1 and Fkbp51 from excised E0771 tumors following normal diet or HFD and treatment with dexamethasone. Data points are mean ± SEM. n ≥ 3. *p < .05; **p < .01 (one-way ANOVA or two-tailed Student's t-test). SSC, side scatter final approval of the manuscript); OF; (data acquisition, statistical analysis of data, data interpretation, and final approval of the manuscript); DM (data acquisition); TR (conception and final approval of the manuscript).

All in vivo experiments were completed under HPRA project license P045, Individual license number I181 and complied with ARRIVE guidelines.

The data that support the findings of this study is available on request.

Stephanie Annett https://orcid.org/0000-0002-5932-251X

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