Mechanisms of acquired resistance to endocrine therapy in hormone-dependent breast cancer cells

https://doi.org/10.1016/j.jsbmb.2007.05.008Get rights and content

Abstract

Acquired resistance is a major problem limiting the clinical benefit of endocrine therapy. To investigate the mechanisms involved, two in vitro models were developed from MCF-7 cells. Long-term culture of MCF-7 cells in estrogen deprived medium (LTED) mimics aromatase inhibition in patients. Continued exposure of MCF-7 to tamoxifen represents a model of acquired resistance to antiestrogens (TAM-R). Long-term estrogen deprivation results in sustained activation of the ERK MAP kinase and the PI3 kinase/mTOR pathways. Using a novel Ras inhibitor, farnesylthiosalicylic acid (FTS), to achieve dual inhibition of the pathways, we found that the mTOR pathway plays the primary role in mediation of proliferation of LTED cells. In contrast to the LTED model, there is no sustained activation of ERK MAPK but enhanced responsiveness to rapid stimulation induced by E2 and TAM in TAM-R cells. An increased amount of ERα formed complexes with EGFR and c-Src in TAM-R cells, which apparently resulted from extra-nuclear redistribution of ERα. Blockade of c-Src activity drove ERα back to the nucleus and reduced ERα–EGFR interaction. Prolonged blockade of c-Src activity restored sensitivity of TAM-R cells to tamoxifen. Our results suggest that different mechanisms are involved in acquired endocrine resistance and the necessity for individualized treatment of recurrent diseases.

Introduction

The estrogen receptor (ER) is expressed in approximately two thirds of breast carcinomas. Estrogen plays a crucial role in promoting growth of ER positive breast cancer cells because removal of the ovaries in premenopausal patients causes objective tumor regression. Current therapeutic strategies involve blockade of the mitogenic effect of estrogen on breast carcinoma by drugs that inhibit ER activity or reduce estrogen biosynthesis. Tamoxifen was the first agent of targeted therapy for hormone-dependent breast cancer and remains the most commonly used agent in the adjuvant setting and for treatment of advanced disease. In addition to tamoxifen, postmenopausal patients benefit from aromatase inhibitors that reduce the levels of estrogen that are critical for tumor growth. Recent clinical trials suggest that third generation aromatase inhibitors are superior to tamoxifen as first line therapy and are effective in patients with disease relapse after tamoxifen treatment [1], [2]. While patients with hormone-dependent breast cancer initially experience objective tumor regression, the disease ultimately relapses. Preclinical and clinical studies have revealed that up-regulation of growth factor signaling pathways is associated with failure of endocrine therapy and targeting the growth factor signaling pathway has become an emerging therapeutic strategy for treatment of breast cancer.

In most cases, there is no loss of ERα when resistance to endocrine therapy develops. The receptor is apparently functioning because a portion of patients with relapsing disease are responsive to secondary endocrine therapies [3], [4]. Increased ER functionality has been found in breast cancer cells that become resistant to manipulations blocking estrogen action [5], [6]. This has been largely attributed to enhanced transcriptional activity of ERα by cross-talk with up-regulated growth factor pathways [7], [8], [9]. However, the process of adaptation to endocrine therapy varies in hormone-dependent breast cancer cells and the precise mechanisms are far from fully understood.

To investigate the mechanisms underlying failure of endocrine therapy, our laboratory has established two in vitro models using MCF-7 cells. Long-term culture of MCF-7 cells in estrogen deprived medium (LTED) mimics aromatase inhibition in patients. Continued exposure of MCF-7 cells to tamoxifen represents a model of acquired resistance to antiestrogens (TAM-R). We found that MCF-7 cells react differently to these two endocrine manipulations. Here we report possible mechanisms that are involved in the process of adaptation to endocrine therapy and re-growth of hormone dependent breast cancer.

Section snippets

Materials

Farnesylthiosalicylic acid (FTS) was a gift from Drs. Yoel Kloog (Tel-Aviv University, Tel-Aviv, Israel) and Wayne Bardin (Thyreos, New York, NY). Estradiol was from Steraloids (Wilton, NH). LY 294002 and rapamycin were purchased from Sigma (St. Louis, MO). U0126 was obtained from Promega (Madison, WI). c-Src kinase inhibitor PP2 was from Calbiochem (San Diego, CA). EGF was obtained from Collaborative Biomedical Products (Bedford, MA). Sources of antibodies for Western analysis are as follows:

Results

We have demonstrated in previous studies that enhanced activity of growth factor pathways in LTED cells renders the cells hypersensitive to the mitogenic effect of estradiol and dual blockade of the MAPK and PI3K pathways reverses the hypersensitive phenotype [10]. We proposed that inhibition of signal transduction at a nodal point where multiple pathways converge would provide an effective strategy for treatment of breast cancer with acquired endocrine resistance. Farnesylthiosalicylic acid

Discussion

The present study demonstrated that different mechanisms are involved when breast cancer cells adapt themselves to escape from the manipulations blocking the estrogen receptor signaling. Long-term estrogen deprivation results in sustained activation of both the MAPK and the mTOR pathways in MCF-7 cells. Using FTS as a tool, we found that the mTOR pathway is a predominant pathway in mediation of proliferation of LTED cells. The specific mTOR inhibitor, rapamycin, effectively inhibited growth of

References (38)

  • B. Long et al.

    Changes in epidermal growth factor receptor expression and response to ligand associated with acquired tamoxifen resistance or oestrogen independence in the ZR-75-1 human breast cancer cell line

    Br. J. Cancer

    (1992)
  • R.I. Nicholson et al.

    Oestrogen and growth factor cross-talk and endocrine insensitivity and acquired resistance in breast cancer

    Br. J. Cancer

    (2000)
  • P. Joel et al.

    pp90rsk1 regulates estrogen receptor-mediated transcription through phosphorylation of Ser-167

    Mol. Cell. Biol.

    (1998)
  • J.M. Gee et al.

    Epidermal growth factor receptor/HER2/insulin-like growth factor receptor signalling and oestrogen receptor activity in clinical breast cancer

    Endocr. Relat. Cancer

    (2005)
  • J. Shou et al.

    Mechanisms of tamoxifen resistance: increased estrogen receptor-HER2/neu cross-talk in ER/HER2-positive breast cancer

    J. Natl. Cancer Inst.

    (2004)
  • W. Yue et al.

    Farnesylthiosalicylic acid blocks mammalian target of rapamycin signaling in breast cancer cells

    Int. J. Cancer

    (2005)
  • N. Pullen et al.

    Phosphorylation and activation of p70s6k by PDK1

    Science

    (1998)
  • C.H. Donovan et al.

    Constitutive MEK/MAPK activation leads to p27Kip1 deregulation and antiestrogen resistance in human breast cancer cells

    J. Biol. Chem.

    (2001)
  • J.M.W. Gee et al.

    Phosphorylation of ERK1/2 mitogen activated protein kinase is associated with poor response to anti-hormonal therapy and decreased patient survival in clinical breast cancer

    Int. J. Cancer

    (2001)
  • Cited by (0)

    View full text