Mechanisms of tumor regression and resistance to estrogen deprivation and fulvestrant in a model of estrogen receptor-positive, HER-2/neu-positive breast cancer.

HER-2/neu in breast cancer is associated with tamoxifen resistance, but little data exist on its interaction with estrogen deprivation or fulvestrant. Here, we used an in vivo xenograft model of estrogen receptor (ER)-positive breast cancer with HER-2/neu overexpression (MCF7/HER-2/neu-18) to investigate mechanisms of growth inhibition and treatment resistance. MCF7/HER-2/neu-18 tumors were growth inhibited by estrogen deprivation and with fulvestrant, but resistance developed in 2 to 3 months. Inhibited tumors had reductions in ER, insulin-like growth factor-I receptor (IGF-IR), phosphorylated HER-2/neu (p-HER-2/neu), and phosphorylated p42/44 mitogen-activated protein kinase (p-MAPK). p27 was increased especially in tumors sensitive to estrogen deprivation. Tumors with acquired resistance to these therapies had complete loss of ER, increased p-HER-2/neu, increased p-MAPK, and reduced p27. In contrast, IGF-IR and phosphorylated AKT (p-AKT) levels were markedly reduced in these resistant tumors. The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib, which can block EGFR/HER-2/neu signaling, significantly delayed the emergence of resistance to both estrogen deprivation and fulvestrant. Levels of p-MAPK and p-AKT decreased with gefitinib, whereas high ER levels were restored. Eventually, however, tumors progressed in mice treated with gefitinib combined with estrogen deprivation or fulvestrant accompanied again by loss of ER and IGF-IR, increased p-HER-2/neu, high p-MAPK, and now increased p-AKT. Thus, estrogen deprivation and fulvestrant can effectively inhibit HER-2/neu-overexpressing tumors but resistance develops quickly. EGFR/HER-2/neu inhibitors can delay resistance, but reactivation of HER-2/neu and signaling through AKT leads to tumor regrowth. Combining endocrine therapy with EGFR/HER-2/neu inhibitors should be tested in clinical breast cancer, but a more complete blockade of EGFR/HER-2/neu may be optimal.

Increased constitutive activity of PKB/Akt in tamoxifen resistant breast cancer MCF-7 cells.

The tamoxifen-resistant (TAM-R) MCF-7 breast cancer cell line has been used as a model to identify the signalling pathways that enable resistant cancer cells to grow independently of steroid hormones. In TAM-R cells, peptide growth factor signalling pathways appear to be important in modified cell behaviour, growth and survival. The PI3 kinase signalling components Akt1 and Akt2 are expressed at similar levels by both parental wild-type MCF-7 and TAM-R cells, but Akt1 phosphorylation is significantly increased in TAM-R cells grown under basal conditions. High levels of basal Akt, GSK3 alpha / beta and p70S6 kinase phosphorylation are all inhibited by the PI3 kinase inhibitor, LY 294002. The ligands for the EGFR/erbB1 receptor, EGF (epidermal growth factor) and TGF alpha (transforming growth factor- alpha ) demonstrate an increased ability to activate Akt in TAM-R compared with parental MCF-7 cells and it is proposed that the preferred autocrine or paracrine activation of Akt occurs via the erbB heterodimer EGFR/erbB2 in TAM-R cells. Akt phosphorylation is reduced by gefitinib ("Iressa"/ZD1839). The results suggest that the PI3 kinase pathway plays a role in proliferation of TAM-R cells and is important in the increased EGF induced membrane ruffling detected in the resistant cells. Increased Akt1 activation may contribute to the aggressive phenotype of tamoxifen resistant ER (oestrogen receptor) positive breast cancers.

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

BACKGROUND: Patients receiving adjuvant tamoxifen whose tumors express high levels of both HER2/neu (HER2) and the estrogen receptor (ER) coactivator AIB1 often develop tamoxifen resistance. We used a breast cancer model system with high expression of AIB1 and HER2 to investigate the possible mechanisms underlying this resistance. METHODS: MCF-7 breast cancer cells, which express high levels of AIB1, and a tamoxifen-resistant derivative cell line engineered to overexpress HER2 (MCF-7/HER2-18) were treated with estrogen, tamoxifen, epidermal growth factor (EGF), or heregulin in the absence or presence of the EGF receptor (EGFR) tyrosine kinase inhibitor gefitinib. We analyzed phosphorylation of signaling intermediates by immunoblotting, ER transcriptional activity with reporter gene constructs and immunoblot analysis of endogenous gene products, promoter assembly by chromatin immunoprecipitation (ChIP) assay, and tumor cell growth in vitro by anchorage-independent colony formation and in vivo using xenografts in nude mice. RESULTS: MCF-7/HER2-18 tumors were completely growth inhibited by estrogen deprivation but were growth stimulated by tamoxifen. Molecular cross-talk between the ER and HER2 pathways was increased in the MCF-7/HER-2 cells compared with MCF-7 cells, with cross-phosphorylation and activation of both the ER and the EGFR/HER2 receptors, the signaling molecules AKT and ERK 1,2 mitogen-activated protein kinase (MAPK), and AIB1 itself with both estrogen and tamoxifen treatment. Tamoxifen recruited coactivator complexes (ER, AIB1, CBP, p300) to the ER-regulated pS2 gene promoter in MCF-7/HER2-18 cells and corepressor complexes (NCoR, histone deacetylase 3) in MCF-7 cells. Gefitinib pretreatment blocked receptor cross-talk, reestablished corepressor complexes with tamoxifen-bound ER on target gene promoters, eliminated tamoxifen's agonist effects, and restored its antitumor activity both in vitro and in vivo in MCF-7/HER2-18 cells. CONCLUSIONS: Tamoxifen behaves as an estrogen agonist in breast cancer cells that express high levels of AIB1 and HER2, resulting in de novo resistance. Gefitinib's ability to eliminate this cross-talk and to restore tamoxifen's antitumor effects should be tested in the clinic.

Oestrogen receptor-mediated modulation of the EGFR/MAPK pathway in tamoxifen-resistant MCF-7 cells.

Oestrogen receptor (ER) levels are usually maintained on acquisition of tamoxifen resistance in the clinic, however, tumour re-growth is associated with increased expression of epidermal growth factor receptor (EGFR) and activation of the mitogen activated protein kinase (MAPK) pathway. In the present study we have used the ER down-regulator fulvestrant ('Faslodex') to investigate the influence of the ER on growth of a tamoxifen-resistant (TAM-R) human breast cancer cell line. Expression levels of ER mRNA and protein were equivalent in parental wild-type MCF-7 (WT) and TAM-R cells. Fulvestrant eliminated ER protein expression and inhibited proliferation in both cell lines. The growth inhibitory effects of fulvestrant were associated with a decrease in basal EGFR, c-erbB2 and ERK1/2 activity in TAM-R but not WT cells. ER functionality as determined by oestrogen response element (ERE)-luciferase reporter activity and expression of PgR, pS2 and transforming growth factor alpha (TGFalpha) was significantly reduced in TAM-R compared to WT cells and was further decreased by fulvestrant treatment in both cell lines. Epidermal growth factor (EGF) and TGFalpha significantly increased EGFR/MAPK pathway activity in both cell lines. Ligand-induced EGFR/MAPK activation promoted TAM-R cell growth in both the absence and presence of fulvestrant, whereas no proliferative activity was observed under the same conditions in WT cells. These results suggest that the ER modulates EGFR/MAPK signalling efficiency in TAM-R cells possibly through the regulation of TGFalpha availability. This effect may be overcome by the action of exogenous EGFR ligands, which strengthen EGFR/MAPK signalling activity to generate endocrine-insensitive cell growth.

Elevated levels of epidermal growth factor receptor/c-erbB2 heterodimers mediate an autocrine growth regulatory pathway in tamoxifen-resistant MCF-7 cells.

The development of acquired resistance to antihormonal agents in breast cancer is a major therapeutic problem. We have developed a tamoxifen-resistant (TAM-R) MCF-7 breast cancer cell line to investigate the mechanisms behind this condition. Both epidermal growth factor receptor (EGFR) and c-erbB2 mRNA and protein expression were increased in TAM-R compared with wild-type MCF-7 cells, whereas comparable levels of c-erbB3 mRNA and protein were expressed in both cell lines. Under basal conditions, phosphorylated EGFR/c-erbB2, EGFR/c-erbB3 but not c-erbB2/c-erbB3 receptor heterodimers were detected in TAM-R cells in association with increased levels of phosphorylated extracellular-signal regulated kinase 1/2 (ERK1/2). Both cell lines were capable of generating a range of EGFR-specific ligands and increased expression of transforming growth factor alpha was observed in TAM-R cells. Treatment of TAM-R cells with ZD1839 (Iressa) or trastuzumab (Herceptin) blocked c-erbB receptor heterodimer formation and phosphorylation, reduced ERK1/2 activity, and strongly inhibited cell growth. The MAPK kinase inhibitor PD098059 specifically reduced phosphorylated ERK1/2 levels and inhibited TAM-R growth. All three agents abolished ERK1/2 activity in wild-type cells but caused only small reductions in cell proliferation. These results demonstrate that TAM-R MCF-7 cell growth is mediated by the autocrine release and action of an EGFR-specific ligand inducing preferential EGFR/c-erbB2 dimerization and downstream activation of the ERK pathway.

ZD1839 (Iressa): an orally active inhibitor of epidermal growth factor signaling with potential for cancer therapy.

The epidermal growth factor receptor (EGFR) is a promising target for anticancer therapy because of its role in tumor growth, metastasis and angiogenesis, and tumor resistance to chemotherapy and radiotherapy. We have developed a low-molecular-weight EGFR tyrosine kinase inhibitor (EGFR-TKI), ZD1839 (Iressa(2) ). ZD1839, a substituted anilinoquinazoline, is a potent EGFR-TKI (IC(50) = 0.033 micro M) that selectively inhibits EGF-stimulated tumor cell growth (IC(50) = 0.054 micro M) and that blocks EGF-stimulated EGFR autophosphorylation in tumor cells. In studies with mice bearing a range of human tumor-derived xenografts, ZD1839 given p.o. once a day inhibited tumor growth in a dose-dependent manner. The level of expression of EGFR did not determine xenograft tumor sensitivity to ZD1839. Long-term ZD1839 (>3 months) treatment of mice bearing A431 xenografts was well tolerated, and ZD1839 completely inhibited tumor growth and induced regression of established tumors. No drug-resistant tumors appeared during ZD1839 treatment, but some tumors regrew after drug withdrawal. These studies indicate the potential utility of ZD1839 in the treatment of many human tumors and indicate that continuous once-a-day p.o. dosing might be a suitable therapeutic regimen.

Epidermal growth factor receptor tyrosine kinase inhibitors.

Clinical trials of selective small-molecule inhibitors of epidermal growth factor receptor tyrosine kinase activity have shown that these targeted inhibitors of proliferative signal transduction provide well-tolerated antitumour activity in patients. Preclinical pharmacology studies illustrate the potential use of these new cancer therapeutics in combination with chemotherapy, radiotherapy and hormone therapy, and in chemoprevention, in a spectrum of solid human tumours.

Multiple responses to EGF receptor activation and their abrogation by a specific EGF receptor tyrosine kinase inhibitor.

BACKGROUND: Epidermal growth factor receptor (EGF-R) autophosphorylation is essential for its intracellular mitogenic signaling via the MAPK pathway and for interaction in other cellular processes. Inhibition of this activity in tumor cells that predominantly utilise EGF-R therefore offers an alternative approach to therapy. METHODS: The ability of a specific inhibitor of EGF-R tyrosine kinase, ZM 252868, (TKI) to alter various parameters related to growth in DU145 and PC3 cell lines was investigated, by immunocytochemistry, Northern blotting, Western blotting and invasion assays. RESULTS: In DU145 cultures, the total cell population and number of cells in cell cycle decreased in the presence of TKI whilst the apoptotic rate was significantly increased. Reduction in autophosphorylation of the EGF-R, membrane expression of EGF-R, activation of the MAPK, p38, and JNK enzymes and the invasive capacity of DU145 cells was observed in the TKI treated cells. Under the same conditions, PC3 cell growth and EGF-R expression and MAPK activation were not affected. The use of inhibitors of intracellular signaling indicated that the DU145 cells, in contrast to PC3 cells, predominantly utilize EGF-R activation of the MAPK signaling pathway for growth. CONCLUSIONS: In prostatic cancer patients, in whom androgen resistance has developed and whose tumors have upregulated EGF-R for growth, specific TKI's may offer an important therapy option.