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

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.

Figure 1

Diagrammatic representation of the actions of physiologic oestradiol (E2) on the growth of small phase II MCF-7 tamoxifen resistant tumors in ovariectomized athymic mice. A larger tumour will regress with oestrdiol treatment but will eventually display oestrogen-stimulated growth. If tumours are retransplanted into a new generation of ovariectomized athymic mice and treated with oestradiol, tamoxifen will block oestrogen-stimulated tumour growth. First presented in St. Gallen, 1993.

Figure 2

The growth of wild type MCF-7 cells (WS8) and various antihormonally resistant sublines in an oestrogen-free environment. The cells MCF-7:5C and 2A grow spontaneously and could be considered to represent aromatase inhibitor resistant cells. These remain OER positive. In contract, MCF-7F are fulvestrant resistant (MCF-7 cells grown for over a year in an oestrogen-deprived environment containing fulvestrant). These cells grow spontaneously but have no OER.

Figure 3

The action of oestradiol (1nM) on the growth of wild type MCF-7 cells (WS8) or long-term oestrogen-deprived MCF-7 cells (5C and 2A). In Panel A the MCF-7:5C cells undergo rapid apoptosis during the first few days of oestradiol exposure whereas the MCF-7:2A cells slowly initiate apoptosis during the days after 6 of oestradiol treatment. In panel B MCF07:5C cells respond to fulvestrant (1μM) with a G1 blockade at 72 hour whereas oestradiol (1nM) causes massive and complete apoptosis. These results were obtained using flow cytometry.

Figure 3

The action of oestradiol (1nM) on the growth of wild type MCF-7 cells (WS8) or long-term oestrogen-deprived MCF-7 cells (5C and 2A). In Panel A the MCF-7:5C cells undergo rapid apoptosis during the first few days of oestradiol exposure whereas the MCF-7:2A cells slowly initiate apoptosis during the days after 6 of oestradiol treatment. In panel B MCF07:5C cells respond to fulvestrant (1μM) with a G1 blockade at 72 hour whereas oestradiol (1nM) causes massive and complete apoptosis. These results were obtained using flow cytometry.

Figure 4

Oestrogenic regulation of apoptotic genes in long term estrogen-deprived MCF-7:5C and MCF-7:2A breast cancer cells as determined by Affymetrix gene microarrays. For experiment, cells were treated with 1nM oestradiol for 48 hours and total RNA was prepared using the Qiagen Rneasy Mini kit. cRNA was generated, labeled, and hybridized to the Affymetrix Human Genome U133 plus 2l0 arrays containing 54,300 probe sets. Chips were then scanned and analyzed using the Affymetrix Microarray Analysis Suite version 5.0. Assessment of data quality was conducted following default guidelines in the Affymetrix’s GeneChip^® Expression Analysis Data Analysis Fundamentals Training Manual. Global scaling for average signal intensity for all arrays was set to 500. Four biological replicates from each of the two cell lines were arrayed to determine consistent and reproducible patterns of gene expression. The above figure shows that oestradiol treatment caused 3- to 6-fold induction of the proapoptotic genes NOXA, GADD45α, GADD45β, BIM, BAX, BAK and p53 in (A) MCF-7:5C cells but only a 2-fold induction of NOXA, BAX, and BAK in (B) MCF-7:2A cells.

Figure 5

The organization of our Department of Defence Center of Excellence Grant entitled “A New Therapeutic Paradigm for Breast Cancer Exploiting Low-Dose Estrogen-Induced Apoptosis.” The model systems to study the survival and apoptosis induced with oestrogen are used for time course experiments at the Fox Chase Cancer Center. The materials are distributed to Translational Genomics for siRNA analysis or gene array and the Vincent T. Lombardi Cancer Center is involved to conduct proteomics. All results are uploaded into a shared secure web for data processing and target identification by our informatics and biostatistical group. Each laboratory is able to validate emerging pathways and study individual genes of interest. Our program is integrated with a clinical trials program that provides patient samples for validation of apoptotic or survival pathways. We are grateful to our external advisory board of Patient Advocates and professional colleagues for their continuing advice and support.

Figure 6

An anticipated treatment plan for third line endocrine therapy. Patients must have responded and failed two successive antihormonal therapies to be eligible for a course of low dose oestradiol therapy for 3 months. The anticipated response rate is 30% and responding patients will be treated with anastrozole until relapse. Validation of the treatment plan via the Center of Excellence grant (Figure 5) will establish a platform to enhance response rates with apoptotic oestrogen by integrating known inhibitors of tumour survival pathways into the 3 month debulking treatment plan. The overall goal is to increase response rates and maintain patients for longer on antihormonal strategies before chemotherapy is required.