Antioestrogen therapy is a highly effective treatment for patients with oestrogen-receptor (ER)-positive breast cancer, emphasising the central role of oestrogen action in the development and progression of this disease. However, effective antioestrogen treatment is often compromised by acquired endocrine resistance, prompting the need for a greater understanding of the downstream mediators of oestrogen action that may contribute to this effect. Recent studies have demonstrated a critical link between oestrogen's mitogenic effects and cell cycle progression, particularly at the G1 to S transition where key effectors of oestrogen action are c-Myc and cyclin D1, which converge on the activation of cyclin E-cdk2. These components are rapidly upregulated in response to oestrogen, and can mimic its actions on cell cycle progression, including re-initiating cell proliferation in antioestrogen-arrested cells. Here we review the roles of c-Myc, cyclin D1 and cyclin E in oestrogen action and endocrine resistance, and identify their potential as markers of disease progression and endocrine responsiveness, and as novel therapeutic targets in endocrine-resistant breast cancer.Endocrine-Related Cancer (2005) 12 S47-S59

We report that transfection of insulin-like growth factor-binding protein-3 (IGFBP-3) cDNA in human breast cancer cell lines expressing either mutant p53 (T47D) or wild-type p53 (MCF-7) induces apoptosis. IGFBP-3 also increases the ratio of pro-apoptotic to anti-apoptotic members of the Bcl-2 family. In MCF-7, an increase in Bad and Bax protein expression and a decrease in Bcl-x L protein and Bcl-2 protein and mRNA were observed. In T47D, Bax and Bad proteins were up-regulated; Bcl-2 protein is undetectable in these cells. As T47D expresses mutant p53 protein, these modulations of pro-apoptotic proteins and induction of apoptosis are independent of p53. The effect of IGFBP-3 on the response of T47D to ionizing radiation (IR) was examined. These cells do not G 1 arrest in response to IR and are relatively radioresistant. Transfection of IGFBP-3 increased the radiosensitivity of T47D and increased IR-induced apoptosis but did not effect a rapid G 1 arrest. IR also caused a much greater increase in Bax protein in IGFBP-3 transfectants compared with vector controls. Thus, IGFBP-3 increases the expression of pro-apoptotic proteins and apoptosis both basally and in response to IR, suggesting it may be a p53-independent effector of apoptosis in breast cancer cells via its modulation of the Bax:Bcl-2 protein ratio.

The role of insulin-like growth factor-binding protein (IGFBP)-5 in human breast cancer cell growth is unclear. We determined the effects of IGFBP-5 expression on the growth of human breast cancer cell lines in vivo and in vitro. Expression of IGFBP-5, both by stable transfection and adenoviral-mediated infection, was inhibitory to the growth of MDA-MB-231 and Hs578T human breast cancer cells over a 13-day period. IGFBP-5 expression resulted in a G 2 /M cell cycle arrest and the induction of apoptosis in both cell lines, an effect that was abrogated in the presence of the broad-spectrum caspase inhibitor, z-VAD-fmk. IGFBP-5-induced apoptosis was associated with a transcriptional increase in expression of the proapoptotic regulator bax and decrease in the anti-apoptotic bcl-2 compared with vector controls. Secreted IGFBP-5 when added exogenously to breast cancer cells was not internalized and had no effect on cell growth or apoptosis, suggesting that IGFBP-5 may elicit its inhibitory effects via a novel, intracrine mechanism. In athymic nude mice, stable expression of IGFBP-5 significantly inhibited both the formation and growth of tumors derived from MDA-MB-231 cells. IGFBP-5-expressing tumors also had a significantly elevated level of bax mRNA and decreased levels of bcl-2 mRNA compared with vector tumors. These data suggest that IGFBP-5 is a potent growth inhibitor and proapoptotic agent in human breast cancer cells via modulation of cell cycle regulation and apoptotic mediators.The insulin-like growth factor-binding proteins (IGFBPs) 1 are a family of proteins that bind with high affinity to IGFs and modulate their mitogenic actions by regulating their ability to interact with their signaling receptor, the type I IGF receptor (IGFRI, reviewed in Ref. 1). However, it is now becoming clear that many IGFBPs have direct roles in the regulation of cell growth and cell death. For example, we and others have demonstrated the antiproliferative and proapoptotic effects of IGFBP-3 in breast (2-4) and prostate (4) cancer cell lines.The role of IGFBP-5 in cell growth is complex, with reports that it can either stimulate or inhibit cell proliferation in various experimental systems (5-7). There is also evidence that IGFBP-5 expression is up-regulated by antiproliferative agents such as retinoic acid (8), vitamin D-related compounds (9), and antiestrogen ICI 182780 (10), with some evidence that it may mediate their growth inhibitory effects (8, 10). Similarly, growth stimulation of human breast cancer cells by estradiol is associated with a down-regulation of IGFBP-5 expression (10), although exogenous IGFBP-5 had no effect on IGF-I-stimulated DNA synthesis in the breast cancer cell line, MCF-7 (11). They demonstrated that addition of exogenous IGFBP-5 to Hs578T cells protects these cells from ceramide-induced apoptosis, suggesting IGFBP-5 may have a survival function in response to apoptotic stimuli (20,21). A similar conclusion was reached by Roschier et al. (22) following their demonstration that induction of apoptosis...

Nedd2 (caspase-2) is a cysteine protease of the caspase family that has been demonstrated to play a role in the apoptotic pathway. The 51-kDa precursor of Nedd2 undergoes cleavage into two subunits following various apoptotic stimuli. In this study, we have investigated the dimerization of the Nedd2 precursor (pro-Nedd2) in Saccharomyces cerevisiae and its self-processing activity in vivo. We demonstrate that the expression of proNedd2 in yeast cells results in processing of the precursor. A catalytically inactive pro-Nedd2 mutant dimerized in yeast, and the dimerization required both the prodomain and the carboxyl-terminal residues. Aspartate mutants that block the removal of the p14/p12 subunits, but not the wild-type Nedd2, were shown to dimerize in yeast cells, suggesting that dimerization occurs prior to processing. In vitro processing of proNedd2 by recombinant active Nedd2 defined the aspartate residues that are crucial for processing to occur. Both the in vivo and in vitro processing of pro-Nedd2 directly correlated with its ability to induce cell death in transient overexpression experiments.

The ICE/CED-3 family of proteases (caspases) play a central role in the execution phase of apoptosis. These proteases are synthesised as precursor molecules that require processing at specific aspartate residues to produce the two subunits that comprise the active enzyme. The activation of some of these proteases has been shown to occur during apoptosis. Here we show that Nedd2/ICH-1 (caspase-2) is activated during apoptosis induced by a variety of apoptotic stimuli. This activation occurs very early upon treatment of cells with apoptotic agents and appears to precede the activation of CPP32 (caspase-3). The activation of Nedd2 was not seen in cells that are resistant to apoptosis. These observations suggest that Nedd2 is an early effector in the pathway leading to cell death. Our observations also lend weight to the hypothesis that a group of caspases containing long prodomains are the first to be activated in response to apoptotic signals and that they lie upstream of a second class of caspases such as CPP32 containing short or no prodomains.

Summary Insulin-like growth factors (IGF) are mitogenic peptides that have been implicated as positive regulators of cellular proliferation. In recent years, several studies have suggested an additional role for the IGF axis in the regulation of apoptosis. Signalling through the IGF receptor has been shown to have a potent survival function and protect cells from a variety of apoptotic stimuli. The actions of IGF are regulated by a family of highaffinity IGF binding proteins (IGFBP), which sequester the IGF from the IGF receptor. However, there is some evidence that one of these binding proteins, IGFBP-3, may have its own pro-apoptotic effects that are independent of its ability to modulate IGF bioavailability. In addition, it has been suggested that the tumour suppressor p53, a crucial mediator of apoptosis in response to cellular stress, may elicit several of its apoptotic effects through manipulation of components of the IGF axis. This review summarizes what is currently known about the role of the IGF system in the regulation of apoptosis, highlighting its implications in the context of tumorigenesis.

BackgroundEstrogen is a pivotal regulator of cell proliferation in the normal breast and breast cancer. Endocrine therapies targeting the estrogen receptor are effective in breast cancer, but their success is limited by intrinsic and acquired resistance.Methodology/Principal FindingsWith the goal of gaining mechanistic insights into estrogen action and endocrine resistance, we classified estrogen-regulated genes by function, and determined the relationship between functionally-related genesets and the response to tamoxifen in breast cancer patients. Estrogen-responsive genes were identified by transcript profiling of MCF-7 breast cancer cells. Pathway analysis based on functional annotation of these estrogen-regulated genes identified gene signatures with known or predicted roles in cell cycle control, cell growth (i.e. ribosome biogenesis and protein synthesis), cell death/survival signaling and transcriptional regulation. Since inducible expression of c-Myc in antiestrogen-arrested cells can recapitulate many of the effects of estrogen on molecular endpoints related to cell cycle progression, the estrogen-regulated genes that were also targets of c-Myc were identified using cells inducibly expressing c-Myc. Selected genes classified as estrogen and c-Myc targets displayed similar levels of regulation by estrogen and c-Myc and were not estrogen-regulated in the presence of siMyc. Genes regulated by c-Myc accounted for 50% of all acutely estrogen-regulated genes but comprised 85% (110/129 genes) in the cell growth signature. siRNA-mediated inhibition of c-Myc induction impaired estrogen regulation of ribosome biogenesis and protein synthesis, consistent with the prediction that estrogen regulates cell growth principally via c-Myc. The ‘cell cycle’, ‘cell growth’ and ‘cell death’ gene signatures each identified patients with an attenuated response in a cohort of 246 tamoxifen-treated patients. In multivariate analysis the cell death signature was predictive independent of the cell cycle and cell growth signatures.Conclusions/SignificanceThese functionally-based gene signatures can stratify patients treated with tamoxifen into groups with differing outcome, and potentially identify distinct mechanisms of tamoxifen resistance.

This study explores the relationship between anti-proliferative signaling by transforming growth factor-␤ (TGF-␤) and insulin-like growth factor-binding protein-3 (IGFBP-3) in human breast cancer cells. In MCF-7 cells, the expression of recombinant IGFBP-3 inhibited proliferation and sensitized the cells to further inhibition by TGF-␤1. To investigate the mechanism, we used T47D cells that lack type II TGF-␤ receptor (TGF-␤RII) and are insensitive to TGF-␤1. After introducing the TGF-␤RII by transfection, the basal proliferation rate was significantly decreased. Exogenous TGF-␤1 caused no further growth inhibition, but immunoneutralization of endogenous TGF-␤1 restored the proliferation rate almost to the control level. The addition of IGFBP-3 did not inhibit the proliferation of control cells but caused dose-dependent inhibition in TGF-␤RII-expressing cells when exogenous TGF-␤1 was also present. Similarly, receptor-expressing cells showed dose-dependent sensitivity to exogenous TGF-␤1 only in the presence of exogenous IGFBP-3. This indicates that in these cells, antiproliferative signaling by exogenous IGFBP-3 requires both the TGF-␤RII and exogenous TGF-␤1. To investigate this synergism, the phosphorylation of TGF-␤ signaling intermediates, Smad2 and Smad3, was measured. Phosphorylation of each Smad was stimulated by TGF-␤1 and, independently, by IGFBP-3 with the two agents together showing a cumulative effect. These data suggest that IGFBP-3 inhibitory signaling requires an active TGF-␤ signaling pathway and implicate Smad2 and Smad3 in IGFBP-3 signal transduction.