Original article
Exploiting the apoptotic actions of oestrogen to reverse antihormonal drug resistance in oestrogen receptor positive breast cancer patients

https://doi.org/10.1016/j.breast.2007.07.020Get rights and content

Abstract

The ubiquitous application of selective oestrogen receptor modulators (SERMs) and aromatase inhibitors for the treatment and prevention of breast cancer has created a significant advance in patient care. However, the consequence of prolonged treatment with antihormonal therapy is the development of drug resistance. Nevertheless, the systematic description of models of drug resistance to SERMs and aromatase inhibitors has resulted in the discovery of a vulnerability in tumour homeostasis that can be exploited to improve patient care. Drug resistance to antihormones evolves, so that eventually the cells change to create novel signal transduction pathways for enhanced oestrogen (GPR30+OER) sensitivity, a reduction in progesterone receptor production and an increased metastatic potential. Most importantly, antihormone resistant breast cancer cells adapt with an ability to undergo apoptosis with low concentrations of oestrogen. The oestrogen destroys antihormone resistant cells and reactivates sensitivity to prolonged antihormonal therapy. We have initiated a major collaborative program of genomics and proteomics to use our laboratory models to map the mechanism of subcellular survival and apoptosis in breast cancer. The laboratory program is integrated with a clinical program that seeks to determine the minimum dose of oestrogen necessary to create objective responses in patients who have succeeded and failed two consecutive antihormonal therapies. Once our program is complete, the new knowledge will be available to translate to clinical care for the long-term maintenance of patients on antihormone therapy.

Introduction

The translation and application of long-term antihormonal strategies, aimed at the tumour oestrogen receptor (OER), has significantly improved the prognosis of patients with breast cancer.1 Long-term adjuvant tamoxifen treatment not only enhances survival and disease-free survival in patients with OER positive tumours during treatment but also reduces mortality for at least 10 years after treatment has stopped.2, 3 Building on the success of long-term tamoxifen therapy, a number of aromatase inhibitors have been shown to improve prognosis and reduce side effects (blood clots and endometrial cancer) if given instead of tamoxifen4, 5, 6 or after tamoxifen treatment.7, 8 Thus, the original scientific strategy9 of long-term antihormonal adjuvant therapy targeted to patients with OER positive disease10, 11 has emerged as the standard of care for breast cancer patients worldwide.

The new dimension of chemoprevention has advanced significantly during the past decade.12 Preliminary studies were initiated in the 1980s to explore the safety and suitability of administering tamoxifen to women only at risk for breast cancer.13, 14, 15 The rationale of these studies was based on the wide clinical experience using tamoxifen to treat all stages of breast cancer, the reduction of contralateral breast cancer noted in patients receiving adjuvant tamoxifen treatment16, 17, 18 and laboratory studies that repeatedly demonstrated that tamoxifen can prevent mammary cancer in animal models.19, 20, 21, 22

The current status and results of the worldwide efforts to quantitate and evaluate the value of tamoxifen as a chemopreventive have been summarized recently23 but it is the P-1 trial completed by Fisher and the National Surgical Adjuvant Breast and Bowel Project (NSABP)24, 25 that is considered to be the landmark.26 The results can be summarized simply. Tamoxifen reduced the incidence of breast cancer by 50%24 in pre and postmenopausal women at high risk.27 Side effects noted were increases in early stage low grade endometrial cancer, blood clots, and cataracts24, 25 but only in postmenopausal women receiving long-term tamoxifen treatment. Tamoxifen is available in the United States for risk reduction in pre and postmenopausal women. However, the consensus today is that tamoxifen is better deployed as a chemopreventive for premenopausal women to reduce the risk of OER positive breast cancer.28, 29, 30, 31, 32 There are no increases in the side effects of endometrial cancer or blood clots but tamoxifen keeps preventing breast cancer long after treatment stops31 consistent with earlier treatment results.3

The concern that tamoxifen was going to be associated with the risk of endometrial cancer33 and the recognition that the drugs called nonsteroidal antioestrogens34 were in fact selective OER modulators (SERMs) led to a paradigm change for chemoprevention. SERMs were oestrogenic in ovariectomized rat bone35 but at the same time prevented mammary cancer.21 These data led to the evidence-based hypothesis that SERMs could prevent breast cancer as a beneficial side effect during the treatment and prevention of osteoporosis.36, 37 Based on this laboratory-based hypothesis, raloxifene was subsequently shown to reduce fractures in postmenopausal women with or at high risk for osteoporosis38 but at the same time caused a 75% reduction in the incidence of breast cancer.39 A follow-up trial P-2 by the NSABP40 established that raloxifene was equivalent to tamoxifen at preventing invasive breast cancer in high risk postmenopausal women but with significantly fewer side effects (hysterectomies, cataracts, overall thrombolic events). However, although lower numbers of endometrial cancer were noted in raloxifene treated women compared to tamoxifen treated women, this was not significant because of a higher hysterectomy rate.40 Nevertheless, a related trial called Raloxifene use for the Heart or RUTH, showed no increase in endometrial cancers during raloxifene treatment compared to placebo arm.41

Thus from this brief introduction, it can be appreciated that significant clinical advances have been made through the application of the principle of long-term antihormone therapy9, 36 for the treatment and prevention of breast cancer. All of the advances can now be applied in clinical practice to improve patient care. Nevertheless, despite these advances through the use of sustained administration of antihormonal drugs, there are consequences for the tumour with the eventual development of drug resistance. In the case of SERMs, the type of resistance is unique and is expressed as SERM stimulated growth.42 But, it is the consistent study of the process of drug resistance to antihormones that resulted in the discovery43 of a weakness in the mechanisms of antihormonal drug resistance that has potential for the future exploitation in clinical practice.

During the past 20 years we have focused our laboratory research program on developing models of SERM resistance in vivo to replicate events that could potentially occur clinically. The models were initially developed in vivo to avoid problems with cell culture where cells that become resistant to short term SERM treatment do not develop the essential requirements for angiogenesis that are necessary to survive and grow in patients. We now have a range of models that have been evaluated for growth in vivo (athymic mice) and that have been passaged in vivo for more than 5–10 years to replicate the long-term antihormonal therapy routinely used to treat patients (Table 1).

Initial studies of resistance to tamoxifen treatment demonstrated the unique feature of SERM stimulated growth. Resistant tumours that develop in athymic mice from both OER positive breast and endometrial cells grow in response to either a SERM or estradiol.33, 44 This is why an aromatase inhibitor or the pure antioestrogen fulvestrant (that binds to OER and facilitates the rapid destruction of the complex)45 are successful second line therapies.46, 47 This form of resistance is referred to as Phase I resistance.42

However, these models represent only a few years of SERM treatment which is inconsistent with clinical experience of 5 years of adjuvant tamoxifen or possibly 10 years or more of raloxifene treatment to maintain bone density. The discovery that long-term SERM treatment exposes a vulnerability in the cancer cell that could have potential therapeutic applications was first reported at the St. Gallen meeting in the early 1990s.43 Simply stated, long-term SERM treatment creates an absolute dependency on the SERM for tumour growth but small physiologic doses of oestradiol cause tumour cell death. Small tumours respond more readily to the apoptotic action of oestrogen but when tumours regrow during continuous oestrogen therapy, the tumours again respond to the SERM or no treatment48 (equivalent to treatment with an aromatase inhibitor for patients). This form of resistance is referred to as Phase II resistance.42 The models for SERM resistance are summarized in Table 1. Thus, it is plausible to consider a clinical strategy whereby limited duration, low dose oestrogen treatment could be used to purge and destroy Phase II resistant breast cancer cells but then patients could be treated again with antihormonal therapy to control tumour growth. However, a case could be made that the ubiquitous use of tamoxifen is declining and over the next decade the standard of care will be long-term treatment with one of several aromatase inhibitors. The question we have addressed in the laboratory is whether long-term oestrogen deprivation of breast cancer cells will expose the vulnerability to the apoptotic actions of oestrogen.

Section snippets

Resistance of breast cancer to oestrogen deprivation

There are two laboratory approaches to developing models of drug resistance to aromatase inhibitors. The traditional model is to study the impact of oestrogen withdrawal on the growth of OER positive breast cancer cells. In contrast, there is a model in vivo employing athymic mice transplanted with MCF-7 cells stably transfected with the aromatase enzyme. Without oestrogen tumours do not grow but when animals are treated with the enzyme substrate androstenedione to make oestrogen, tumour growth

The new biology of oestrogen action

A re-examination of MCF-7 clones 5C and 2A occurred at the time when clinical investigators were re-examining the value of high dose oestrogen therapy in those patients who had been treated exhaustively with successive antihormonal therapies.64 The clinical studies demonstrated that high dose oestrogen therapy could cause tumour regression or stasis (30%) in patients treated exhaustively with antihormones.64 Additionally, high concentrations of oestrogen could induce apoptosis in long-term

Analysis of apoptotic pathways

A number of U-133 Affymetrix gene arrays were completed using the MCF-7, MCF-7:5C and 2A cell lines to define the early events of oestrogen action. A 48 h time point was used in our preliminary studies and five replicates were analysed to ensure statistical veracity. All gene array analyses were completed at Translational Genomics, AZ. Results illustrated in Fig. 4 show the 48 h increase in proapoptotic genes that are activated by oestrogen in the MCF-7:5C cells. This is consistent with the time

Translation of laboratory results to patient care

We have established a multi-center collaborative translational research grant with headquarters at the Fox Chase Cancer Center (FCCC) (Fig. 5, Fig. 6). The five year program is sponsored by the US Department of Defense Breast Cancer Program BC050277 entitled “A New Therapeutic Paradigm for Breast Cancer Exploiting Low-Dose Estrogen-Induced Apoptosis.”

Our goal is to create maps of the survival and apoptotic responses to oestrogen noted in our models in vivo and in vitro. Biological samples from

Conflict of Interest

None declared.

Acknowledgements

Supported (VCJ) by the Department of Defense Breast Program under award number BC050277 Center of Excellence (Views and opinions of, and endorsements by the author(s) do not reflect those of the US Army or the Department of Defense), SPORE in Breast Cancer CA 89018, R01 GM067156, FCCC Core Grant NIH P30 CA006927, the Avon Foundation and the Weg Fund of Fox Chase Cancer Center. Ramona Swaby, MD is the recipient of the clinical trials grant from AstraZeneca.

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