An in vitro model showing adaptation to long-term oestrogen deprivation highlights the clinical potential for targeting kinase pathways in combination with aromatase inhibition

doi:10.1016/j.steroids.2011.02.035

Steroids

Volume 76, Issue 8, July 2011, Pages 772-776

https://doi.org/10.1016/j.steroids.2011.02.035 Get rights and content

Abstract

Aromatase inhibitors (AI) have improved the treatment of oestrogen receptor positive (ER+) breast cancer. Despite the efficacy of these agents over 40% of patients relapse with endocrine resistant disease. Here we describe an in vitro model of acquired resistance to long-term oestrogen deprivation (LTED). The LTED cells retain expression of the ER and appear hypersensitive to oestrogen as a result of altered kinase activity. Furthermore analysis of temporal changes in gene expression during the acquisition of resistance highlight growth factor receptor pathways as key mediators of this adaptive process.

Introduction

Oestrogen (E) plays a pivotal role in the development of oestrogen receptor positive (ER+) breast cancer. This has been exploited clinically by the development of endocrine therapies. These seek to deprive the hormone dependent tumour cells of E using anti-oestrogens such as tamoxifen and fulvestrant (ICI 182 780) both of which compete with E for the oestrogen receptor (ER) or aromatase inhibitors (AI), which block the conversion of androgens into E [1]. AIs have improved the treatment of ER+ early and advanced breast cancer [2], [3]. However, one of the major hurdles in the clinical management of hormone receptor positive breast cancer is the development of endocrine resistance. Identification of the molecular mechanisms associated with relapse on endocrine therapy is of paramount importance for the identification of new biomarkers together with novel therapeutic targets. In an effort to address this we, like some other groups, have developed cell lines modelling relapse on an AI [4], [5], [6], [7], [8]. In vitro and in vivo studies suggest that cross-talk between the ER and growth factor pathways allow breast tumours to circumvent the need for steroid hormones whilst in most cases retaining expression of the ER [9], [10]. In the following manuscript we review previous data and provide some insight into the temporal changes in gene expression that occur during acquisition of resistance to long-term oestrogen deprivation (LTED).

Section snippets

Materials and methods

For detailed methodologies see Refs. [11], [13].

Results and discussion

During adaptation to LTED the MCF7 cells passed through two distinct phases: quiescent, followed by hypersensitivity where basal cell growth was maximally stimulated by concentration of oestradiol (E2) as low as 10⁻¹³ M (Fig. 1A). In contrast the wt-MCF7 cells required concentrations in excess of 10⁻¹⁰ M [5], [9], [11], [13]. Clinical data supporting hypersensitivity as a means of resistance comes from a study of pre-menopausal patients who initially responded and then relapsed after E withdrawal

Acknowledgements

We thank the Mary-Jean Mitchell Green Foundation and Breakthrough Breast Cancer for funding. We also acknowledge NHS funding to the Royal Marsden NIHR Biomedical Research Centre.

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Cited by (17)

Optimal adjuvant endocrine treatment of ER+/HER2+ breast cancer patients by age at diagnosis: A population-based cohort study
2018, European Journal of Cancer
Citation Excerpt :
Indeed, results from the combined SOFT/TEXT analysis confirmed that the AI exemestane plus ovarian ablation (OA) significantly improved disease-free survival (DFS), breast cancer-free interval and distant metastasis-free survival when compared with TAM plus OA and TAM alone [2,3]. Studies on the crosstalk between the ER and human epidermal growth factor receptor 2 (HER2) pathways, however, resulted in the discovery of a differential endocrine treatment response between ER+/HER2− and ER+/HER2+ preclinical breast cancer models, suggesting that ER+/HER2+ cell lines are resistant to TAM [4–7]. Clinical studies confirmed the benefit of AI over TAM in postmenopausal ER+/HER2+ patients, although the difference was not significant [8–10].
Prior randomised controlled trials on adjuvant hormonal therapy included HER2_any patients; however, a differential effect of aromatase inhibitors (AIs) versus tamoxifen (TAM) may have been missed in ER+/HER2+ patients that comprise 7–15% of all breast cancer patients.
In addition, a woman's hormonal microenvironment may influence sensitivity to TAM and AIs in the adjuvant setting, which changes during menopausal transition, a process that takes years. We studied the efficacy of AIs versus TAM in ER+/HER2+ breast cancer patients grouped by age at diagnosis as a proxy for menopausal status using treatment and outcome data from the nationwide population-based Netherlands Cancer Registry (NCR).
All women diagnosed between 2005 and 2007 with endocrine-treated, T_anyN_anyM₀, ER+/HER2+ breast cancer were identified through the NCR (n = 1155). Patients were divided by age at diagnosis: premenopausal (≤45 years; n = 326), perimenopausal (45<years≤55; n = 304) and postmenopausal (>55 years; n = 525). A time-dependent variable, indicating whether AI or TAM was received for >50% of endocrine treatment duration, was applied to subdivide groups by predominant treatment received. Recurrence-free survival (RFS) and overall survival (OS) were assessed using Kaplan–Meier survival estimation and Cox regression. Hazard ratios (HRs) were adjusted for chemotherapy, trastuzumab, age at diagnosis, N-status, grade, pT-stage and ovarian ablation.
During follow-up, 237 recurrences and 182 deaths occurred. Perimenopausal women derived significant RFS and OS benefit from AI compared with TAM, HR 0.47 (95% CI 0.25–0.91; P = 0.03) and HR 0.37 (95% CI 0.18–0.79; P = 0.01), respectively, whereas premenopausal women derived no benefit from AI compared with TAM. Treatment effects differed significantly between these age groups (interaction P = 0.03 and P = 0.02, respectively). Among postmenopausal women a small but non-significant AI benefit was observed.
AI treatment, preferably without any TAM treatment, was associated with the best RFS and OS outcome in ER+/HER2+ perimenopausal breast cancer patients.
Estrogen can restore Tamoxifen sensitivity in breast cancer cells amidst the complex network of resistance
2017, Biomedicine and Pharmacotherapy
Citation Excerpt :
Despite estrogen having a lower impact on Tam-R cell growth due to reduced ERα66 expression, contrary to responsive cells [28], ERα66 was found to be over-activated in Tam-R cells [14]. Regardless, LTED-R cells are more hypersensitive and reliant on estrogen than Tam-R cells are [29–31]. Therefore, the administration of aromatase inhibitors cannot inhibit LTED-R proliferation[3].
Breast cancer-related deaths have been on the decline ever since the application of systemic therapies. Chiefly, endocrine therapy, such as Tamoxifen, enhances the survival of estrogen receptor (ER)-positive patients. More than a decade has passed since the introduction of Tamoxifen, however, drug resistance, particularly to Tamoxifen, still remains a major challenge. It has been shown that not only does chronic Tamoxifen exposures induce resistance, but estrogen deprivation can as well. There are two Tamoxifen resistant cell lines, long term estrogen deprived (LTED) cells and cells that have acquired resistance due to long-term exposure to Tamoxifen (Tam-R). Despite having similar cytosolic pathways over-activated in Tam-R and LTED-R cells during the development of resistance, the administration of receptor tyrosine kinases (RTKs) inhibitors fail to restore Tamoxifen sensitivity in LTED-Rs. This alludes to existing differences in the underlying molecular mechanisms of resistance. Surprisingly, despite estrogen being recognized as a breast cancer stimulator; it has recently been introduced as an apoptotic inducer in unresponsive cells. Furthermore, the addition of estrogen to the media of LTED and Tam-R cells triggers cell death, perhaps is functioning as an anti-proliferative agent. In this review, we outline the molecular pathways potentially facilitating estrogen-induced apoptosis in resistant cells.
Biomarker changes associated with the development of resistance to aromatase inhibitors (AIs) in estrogen receptor-positive breast cancer
2014, Annals of Oncology
Citation Excerpt :
Moreover, recurrences occur in a sizeable minority of patients treated with adjuvant AIs [2, 3]. Much effort has been expended in trying to elucidate the mechanisms involved in endocrine resistance [4–10]. The discovery of predictive biomarkers has been primarily based on correlative analysis between the molecular characteristics of the primary tumours and time to recurrence, in patients treated with adjuvant [5, 6] or neoadjuvant therapy [7–10].
The purpose of this study was to identify any differences in key biomarkers associated with estrogen action between biopsies taken at diagnosis and at recurrence or progression during treatment with an aromatase inhibitor (AI).
Patients were retrospectively identified from a clinical database as having relapsed or progressed during AI treatment. Immunohistochemistry was carried out against estrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2), insulin-like growth factor type-1 receptor (IGF1R), insulin receptor substrate-1 (IRS-1), stathmin, phosphatase and tensin homolog and Ki67.
Fifty-five pairs of samples were identified with ER- and/or PgR-positive diseases. Four (7%) patients were ER-negative at progression. Overall, PgR levels were lower in the recurrence sample, but 35% of cases remained positive. IGF1R levels decreased significantly. There were no substantial changes in HER2, IRS-1 or stathmin levels to indicate a role in resistance. Higher Ki67 levels at resistance indicate more proliferative disease.
The phenotype of AI-recurrent lesions shows high between-tumour heterogeneity. There is evidence of an increase in Ki67, a reduction in IGF1R and a loss of ER expression in some individuals and some activation of growth factor signalling pathways that may explain resistance in individuals and merit treatment targeted to those pathways. Biopsy at recurrence will be necessary to identify the relevant target for individuals.
Candidate methylation sites associated with endocrine therapy resistance in ER+/HER2- breast cancer
2020, BMC Cancer
Single-cell transcriptomics reveals multi-step adaptations to endocrine therapy
2019, Nature Communications
New insights into acquired endocrine resistance of breast cancer
2019, Cancer Drug Resistance

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An in vitro model showing adaptation to long-term oestrogen deprivation highlights the clinical potential for targeting kinase pathways in combination with aromatase inhibition

Abstract

Introduction

Section snippets

Materials and methods

Results and discussion

Acknowledgements

Lancet

J Steroid Biochem Mol Biol

J Biol Chem

J Steroid Biochem Mol Biol

Breast cancer: aromatase inhibitors take on tamoxifen

Nat Med

Randomized trial of letrozole following tamoxifen as extended adjuvant therapy in receptor-positive breast cancer: updated findings from NCIC CTG MA.17.nJ

Natl Cancer Inst

Estrogen deprivation causes estradiol hypersensitivity in human breast cancer cells

J Clin Endocrinol Metab

Elevated mitogen-activated protein kinase activity in estrogen-nonresponsive human breast cancer cells

Cancer Res