UPDATE ON BREAST CANCER: EVIDENCE LINKS ESTROGEN, ERBB2, AND AKT

Natasha Kasid, Mary Beth Martin, Ph.D., and Adriana Stoica, Ph.D.

Department of Human Science, School of Nursing and Health Studies, and Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, D.C. 20007.

Keywords: Breast Cancer, Estrogen, Growth Factors, ErbB2, Estrogen Receptor-?, AKT.

Introduction
Admission of breast cancer patients to intensive care units is not uncommon and metastatic disease associated with major organ system failure is the major reason (Headley et al., 1992; Comley et al., 1999; Huaringa et al., 2000; Haas, 2003; Buchheidt et al., 2004; Soares et al. 2004). Patients with breast cancer may develop malignancy-related pericardial effusion (Wang et al., 2002), hypercalcemia (Sculier, Markiewicz, 1991), acute respiratory failure during chemotherapy (Ben-Abraham et al, 1997), postoperative thromboembolism (Agnelli, 1997; Goldhaber, 1998) and disseminated intravascular coagulation (Koga et al., 2004), or infections due to the use of aggressive treatments (Buchheidt et al., 2004). Tamoxifen, an anti-estrogen, has been shown to increase venous thromboembolism (VTE) in postmenopausal women with breast cancer (Grady et al., 2000). In a breast cancer prevention trial, women at risk for atherosclerosis had a higher risk of VTE during tamoxifen therapy (Decensi et al., 2005).

Estrogen receptor-? (ER- ?) is a well-known target of estrogen in hormone-stimulated breast cancer (Tsai and O’Malley, 1994; Kato et al., 1998). Indeed, ER-? expression in breast tumor is a selection factor for anti-estrogen therapy. The ErbB2/HER-2/Neu receptor is overexpressed in approximately 15-20% of breast cancers, and a humanized monoclonal antibody targeting HER-2 (Herceptin/Trastuzumab) is widely used for the treatment of breast cancer patients (Slamon et al., 1987; Carter et al., 1992). However, transtuzumab therapy is limited to patients with very high levels of ErbB2/HER-2 and is associated with cardiac toxicity (Albanell et al., 2003). A third molecule, AKT plays an important role in cell survival, proliferation and metabolism (Thompson and Thompson, 2004). The purpose of this update is to briefly review recent laboratory data showing a correlation between estrogen, ER-?, ErbB2, and AKT. In addition, growth factors such as insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), and heregulin-?1 (HRG-?1) have been shown to activate AKT and ER-? via their cognate receptors, IGFR-I, EGFR, and ErbB3, respectively (Martin et al., 2000). Knowledge of the estrogen and growth factor-stimulated AKT pathway(s) could lead to a molecular target in breast cancer therapy and improved care of the critically ill cancer patients.

Estrogen, estrogen receptor-?, and breast cancer

Numerous studies have shown that estrogen (17?-estradiol), a reproductive steroid hormone, plays an important role in initiation and progression of breast cancer (Parl, 2000). The biological effects of estrogen are mediated by binding to estrogen receptor (ER), a nuclear phosphoprotein and a transcription factor. The activated ER interacts with estrogen response elements in the promoter region of target genes involved in cell growth and proliferation (Kato et al., 1998). It is not surprising that exposure of ER-positive breast tumor cells to estrogen enhances cell growth and proliferation. An anti-estrogen, e.g. tamoxifen, can bind to ER and block the effects of estrogen on breast epithelial cells (Early Breast Cancer Trialists’ Collaborative Group, 1998; Robertson, 2004). ER-?, a well-studied isoform of ER, in breast tissue mediates the effects of estrogen and its activation may increase the risk for breast cancer. In post-menopausal women, hormone replacement therapy, aimed at preventing the loss of bone mineral density (BMD), increases the risk for ER-positive breast cancer. The Nurses’ Health Study reported a high incidence of ER positive breast tumors in patients receiving post-menopausal hormone therapy (Colditz et al., 2004; Chen et al., 2004). These observations demonstrate that ER is a clinically relevant biomarker of breast cancer, and highlight the importance of further elucidation of mechanisms of estrogen-induced breast cancer.

Growth factors, receptor tyrosine kinases, and AKT

ER-positive breast cancer cells respond to a number of growth factors. These factors are secreted by the tumor cells and affect tumor cell proliferation in an autocrine or paracrine manner. In general, the growth factor binds to its receptor tyrosine kinase on the cell surface, resulting in the activation of the receptor, followed by a series of phosphorylation and dephosphorylation signals that ultimately cause changes in gene expression. One important growth factor-induced molecular signaling pathway is known as the phosphatidylinositol 3-kinase (PI 3-kinase) and AKT pathway (Thompson and Thompson, 2004). PI3-Kinase is a membrane-bound lipid kinase, and AKT, also known as protein kinase B (PKB), is a cytosolic serine-threonine kinase and a cancer-causing gene (oncogene). Growth factor stimulation of a transmembrane receptor tyrosine kinase, for example, EGF receptor/ErbB1, ErbB2/HER2/Neu, or IGF-I receptor leads to activation of PI3-kinase. Activated PI3-kinase phosphorylates phosphatidylinositol diphosphate (PIP-2), a membrane lipid, to form phosphatidylinositol triphosphate (PIP-3). PIP-3, in turn, binds to AKT and recruits it to the membrane where it is phosphorylated and activated by a membrane-bound protein serine-threonine kinase, PDK1. Molecular and biological effects of activated AKT are numerous and include: 1) inactivation of Bad, a cell death inducer, 2) activation of mTOR, a serine-threonine kinase associated with increased glucose uptake and glycolysis, and 3) activation of ER-? and changes in gene expression as discussed below.

Estrogen rapidly stimulates AKT (Non-genomic estrogen actions)

Treatment of ER-? positive breast cancer cells with estrogen causes rapid interaction of ER-? with PI3-kinase and activation of AKT (Ahmad et al., 1999; Simoncini et al., 2000; Tsai et al., 2001; Stoica et al., 2003). In this situation, estrogen binds to the non-nuclear (membrane-associated) pool of ER-?. The key observations are summarized below.

• In ER-positive breast cancer cells, estrogen activates AKT1, an isoform of AKT. Two receptors, ErbB2 and non-nuclear ER-? are required in this process.
• In ER-negative breast cancer cells that express ErbB2, estrogen activates AKT3, a different isoform of AKT.

Growth factors increase estrogen receptor-? activity via AKT

The anti-estrogen therapy has been the treatment modality for most ER-positive breast cancer patients. However, not all ER-positive breast cancers respond to anti-estrogens. Tamoxifen resistance is heterogeneous and multifactorial, one mechanism being tamoxifen stimulation of tumor growth. Other changes may occur at the level of the target ER-?, at a post-receptor point in the ER-? response pathway and/or downstream of this pathway, or in growth factor-induced ER-? activity through activation of protein kinases resulting in phosphorylation of ER-? (Osborne et al., 2002). ER-positive breast cancer cells secrete a variety of growth factors which may modulate their growth and proliferation. We have demonstrated growth factor-induced activation of ER-? in these breast cancer cells (Martin et al., 2000). Other reports have shown that AKT phosphorylates ER-?, and AKT expression correlates with increased expression of estrogen/ER-?-regulated pS2 gene in MCF-7 cells (Campbell et al., 2001). Our data are summarized below.

• Growth factors (IGF-I, heregulin and EGF) induce the mRNA expression of ER-?-inducible progesterone receptor and pS2 genes in MCF-7 breast cancer cells. The mRNA induction is blocked in the presence of antiestrogen (ICI 182,780) or wortmannin, an inhibitor of PI3-K. Increased phosphorylation of ER-? also occurs in the presence of an activated mutant of AKT. These results suggest that IGF-I and EGF stimulate ER-? activity via the PI3-kinase/AKT pathway (Martin et al., 2000; Stoica et al., 2000c).

Summary and clinical relevance

• AKT plays an important role in estrogen and growth factor-induced activation of ER-? in breast cancer cells.
• ErbB2 is a component of the estrogen-induced AKT pathway.
• AKT may confer resistance of ER-positive (and ErbB2-positive) breast cancer to tamoxifen therapy.
• Combination of an inhibitor of ErbB2, AKT and an anti-estrogen at lower doses that used in monotherapies could be beneficial in ER-positive breast cancers with low levels of ErbB2, but active AKT.

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