Katrine Sonne-Hansen

Ida C Norrie

Kristina B Emdal

Rikke V Benjaminsen

Thomas Frogne

Ib J Christiansen

Tove Kirkegaard

Anne E Lykkesfeldt

Ida C Norrie Ae

Kristina B Ae

A E Emdal

V Rikke

A E Benjaminsen

Frogne Thomas

Ib J Ae

A E Christiansen

Kirkegaard Tove

Anne E Ae

Lykkesfeldt

Á K B Emdal

Á R V Benjaminsen

Á T Frogne

Á T Kirkegaard

Á A E Lykkesfeldt

Breast cancer cells can switch between estrogen receptor α and ErbB signaling and combined treatment against both signaling pathways postpones development of resistance

Keywords: Breast cancer, Treatment resistance, ERa, ErbB, Fulvestrant, CI-1033, Combined treatment

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The antiestrogen tamoxifen has been first line endocrine treatment for ERa positive breast cancer patients for more than two decades. After an initial response in the majority of patients, almost all patients with advanced disease develop resistance to the therapy. Treatment with the pure antiestrogen fulvestrant has proven effective upon progression on tamoxifen both in vitro and in vivo and is approved for second line endocrine treatment [START_REF] Bru ¨nner | MCF7/LCC2: a 4-hydroxytamoxifen resistant human breast cancer variant that retains sensitivity to the steroidal antiestrogen ICI 182, 780[END_REF][START_REF] Lykkesfeldt | Altered expression of estrogen-regulated genes in a tamoxifen-resistant and ICI 164, 384 and ICI 182, 780 sensitive human breast cancer cell line, MCF-7/TAMR-1[END_REF][START_REF] Howell | Response to a specific antioestrogen (ICI 182780) in tamoxifenresistant breast cancer[END_REF][START_REF] Osborne | Fulvestrant: an oestrogen receptor antagonist with a novel mechanism of action[END_REF]. However, as for treatment with tamoxifen, development of resistance will inevitably occur. The molecular mechanisms contributing to the development of antiestrogen resistance are still elusive, but increasing evidence suggests that aberrant activation of growth factor signaling cascades can provide proliferation and survival signals, overcoming the effect of antiestrogen treatment. Several preclinical studies of fulvestrant resistance have reported changes in the ErbB system, consisting of the EGF receptor (EGFR/ HER1), ErbB2 (HER2/neu), ErbB3 (HER3), and ErbB4 (HER4) receptors and at least 12 activating ligands. The changes correlated with fulvestrant resistance have been observed at the ErbB receptor level, the ligand level, and in ErbB downstream signaling molecules [START_REF] Mcclelland | Enhanced epidermal growth factor receptor signaling in MCF7 breast cancer cells after long-term culture in the presence of the pure antiestrogen ICI 182, 780 (Faslodex)[END_REF][START_REF] Atlas | Heregulin is sufficient for the promotion of tumorigenicity and metastasis of breast cancer cells in vivo[END_REF][START_REF] Sommer | Studies on the development of resistance to the pure antiestrogen faslodex in three human breast cancer cell lines[END_REF][START_REF] Frogne | Antiestrogen-resistant human breast cancer cells require activated protein kinase B/Akt for growth[END_REF][START_REF] Massarweh | Mechanisms of tumor regression and resistance to estrogen deprivation and fulvestrant in a model of estrogen receptor-positive, HER-2/neu-positive breast cancer[END_REF][START_REF] Fan | Diverse gene expression and DNA methylation profiles correlate with differential adaptation of breast cancer cells to the antiestrogens tamoxifen and fulvestrant[END_REF][START_REF] Frogne | Activation of ErbB3, EGFR and Erk is essential for growth of human breast cancer cell lines with acquired resistance to fulvestrant[END_REF]. Consequently, therapies targeting the ErbB system represent an attractive strategy in breast cancer treatment. Focus has primarily been on EGFR and ErbB2 targeted treatment, but such therapies have shown limited clinical efficacy due to inherent or acquired resistance. For trastuzumab targeting the ErbB2 receptor, response rates of approximately 30% have been reported in patients with ErbB2 overexpressing tumors, whereas the benefit of EGFR targeted therapies may be as low as 5-15% (reviewed in [START_REF] Hobday | Molecularly targeted therapies for breast cancer[END_REF]). The limited effects of EGFR and ErbB2 monotherapy could be due to the tight integration and redundancy of the ErbB system in sustaining tumor growth and progression, for example through activation of the ErbB3 receptor (reviewed in [START_REF] Stern | ERBB3/HER3 and ERBB2/HER2 duet in mammary development and breast cancer[END_REF]). Further, the importance of both ErbB and ERa signaling in breast cancer have raised focus on interactions between these receptors and increasing amounts of data suggest that cross-receptor interactions are more common than previously anticipated and may affect growth of breast cancer cells and their response to treatment. For example, ERa may activate membrane tyrosine kinases through nongenomic interactions that may lead to activation of downstream effector molecules, which in turn may exert positive feedback to activate ERa signaling. Such a bidirectional cross-talk can augment signaling of both ERa and ErbB pathways, enhance growth and survival of breast cancer cells and may be involved in tamoxifen resistance [START_REF] Massarweh | Resistance to endocrine therapy in breast cancer: exploiting estrogen receptor/growth factor signaling crosstalk[END_REF][START_REF] Pancholi | ERBB2 influences the subcellular localization of the estrogen receptor in tamoxifenresistant MCF-7 cells leading to the activation of AKT and RPS6KA2[END_REF]. In contrast, tumor cells which escape fulvestrant treatment leading to ERa downregulation are more likely to develop ERa independent upregulation of growth factor receptor signaling. Furthermore, preclinical data have suggested that increased growth factor expression and signaling through the ErbB receptors or downstream effectors may repress ERa expression and function [START_REF] Liu | MCF-7 breast cancer cells overexpressing transfected c-erbB-2 have an in vitro growth advantage in estrogen-depleted conditions and reduced estrogen-dependence and tamoxifen-sensitivity in vivo[END_REF][START_REF] Stoica | Regulation of estrogen receptor-alpha gene expression by epidermal growth factor[END_REF][START_REF] Oh | Hyperactivation of MAPK induces loss of ERalpha expression in breast cancer cells[END_REF][START_REF] Bayliss | Reversal of the estrogen receptor negative phenotype in breast cancer and restoration of antiestrogen response[END_REF], potentially making breast cancer cells less sensitive to endocrine therapy. Also, clinical data have indicated an inverse relationship between expression of at least the EGFR and ErbB2 receptors with ERa and their overexpression has been correlated to decreased antiestrogen sensitivity [START_REF] Houston | Overexpression of c-erbB2 is an independent marker of resistance to endocrine therapy in advanced breast cancer[END_REF][START_REF] Arpino | Estrogen receptor-positive, progesterone receptor-negative breast cancer: association with growth factor receptor expression and tamoxifen resistance[END_REF][START_REF] De Laurentiis | A meta-analysis on the interaction between HER-2 expression and response to endocrine treatment in advanced breast cancer[END_REF][START_REF] Rasmussen | Adjuvant letrozole versus tamoxifen according to centrally-assessed ERBB2 status for postmenopausal women with endocrine-responsive early breast cancer: supplementary results from the BIG 1-98 randomised trial[END_REF].

We have established a model system with acquired fulvestrant resistant breast cancer cell lines developed from parental MCF-7 cells [START_REF] Lykkesfeldt | Human breast cancer cell lines resistant to pure anti-estrogens are sensitive to tamoxifen treatment[END_REF]. These resistant cell lines show increased expression of EGFR and the ErbB3/4 ligand heregulin-2b, increased activation of ErbB3, Erk, and Akt, and decreased expression of ErbB4 [START_REF] Frogne | Activation of ErbB3, EGFR and Erk is essential for growth of human breast cancer cell lines with acquired resistance to fulvestrant[END_REF]. The resistant cell lines were preferentially sensitive to treatment with the tyrosine kinase inhibitor gefitinib targeting EGFR, compared to parental antiestrogen sensitive MCF-7 cells [START_REF] Frogne | Activation of ErbB3, EGFR and Erk is essential for growth of human breast cancer cell lines with acquired resistance to fulvestrant[END_REF]. However, in our cell lines, 1 lM gefitinib did not only inhibit EGFR phosphorylation but also ErbB3 and ErbB4 phosphorylation ( [START_REF] Frogne | Activation of ErbB3, EGFR and Erk is essential for growth of human breast cancer cell lines with acquired resistance to fulvestrant[END_REF] and unpublished data for ErbB4). Thus, the potent effect of gefitinib in our resistant cell lines may be due to the pan-ErbB inhibiting effects. CI-1033 is a novel irreversible pan-ErbB inhibitor that is highly effective in inhibiting the four ErbB receptors and causes prolonged suppression of ErbB kinase activity in breast cancer cells [START_REF] Slichenmyer | CI-1033, a pan-erbB tyrosine kinase inhibitor[END_REF][START_REF] Allen | Potential benefits of the irreversible pan-erbB inhibitor, CI-1033, in the treatment of breast cancer[END_REF]. CI-1033 has been suggested as a promising anticancer drug due to the involvement of ErbB signaling in various cancers. Preclinical data proved the efficacy of CI-1033 in two different breast xenograft models [START_REF] Allen | Potential benefits of the irreversible pan-erbB inhibitor, CI-1033, in the treatment of breast cancer[END_REF]. Furthermore, CI-1033 inhibited ErbB downstream signaling through Akt and Erk and induced apoptosis in BT474 breast cancer cells, whereas CI-1033 alone could not induce apoptosis in MDA-MB-453 cells [START_REF] Nelson | Akt, MAPK (Erk1/2), and p38 act in concert to promote apoptosis in response to ErbB receptor family inhibition[END_REF]. Clinically, CI-1033 has entered phase I and II trials and has demonstrated evidence of target biomarker modulation and antitumor activity [START_REF] Allen | Potential benefits of the irreversible pan-erbB inhibitor, CI-1033, in the treatment of breast cancer[END_REF]. A few heavily pretreated breast cancer patients have been enrolled in phase I studies with CI-1033 and stable disease for more than three months was observed in a small subset of these patients [START_REF] Allen | Potential benefits of the irreversible pan-erbB inhibitor, CI-1033, in the treatment of breast cancer[END_REF][START_REF] Allen | CI-1033, an irreversible pan-erbB receptor inhibitor and its potential application for the treatment of breast cancer[END_REF]. However, a recent phase II trial with CI-1033 in pretreated metastatic breast cancer patients expressing one or more ErbB receptors showed disappointing results [START_REF] Rixe | A randomized, phase II, dosefinding study of the pan-ErbB receptor tyrosine-kinase inhibitor CI-1033 in patients with pretreated metastatic breast cancer[END_REF]. Antitumor activity was only observed at doses reaching unacceptable levels of toxicity. Yet, the study was performed in heavily pretreated patients, which may have limited the effectiveness of CI-1033.

The present study was designed to clarify the importance of ErbB and ERa signaling for growth of breast cancer cells with focus on fulvestrant resistant breast cancer. In a panel of fulvestrant resistant cell lines, we investigated the growth inhibitory effect of treatment with EGFR and ErbB2 neutralizing antibodies and with the pan-ErbB inhibitor CI-1033 alone or in combination with the Akt inhibitor SH-6 and the Protein Kinase C (PKC) inhibitor RO-32-0432. CI-1033 selectively inhibited resistant cell growth compared to growth of parental MCF-7 cells. Further, the effect of CI-1033 was investigated in presence and absence of fulvestrant, which completely blocks ERa signaling. Withdrawal of fulvestrant from the growth medium of fulvestrant resistant cells resulted in reexpression of ERa and significantly reduced the sensitivity to CI-1033 treatment, indicating that the resistant cells are able to switch to ERa signaling upon blockade of ErbB signaling. Concurring, it was tested and found that activation of ErbB signaling with heregulin-1b in parental MCF-7 cells could substitute for repression of ERa-mediated cell growth with fulvestrant. The above-mentioned experiments point toward a high flexibility for breast cancer cells to promote growth through ERa or ErbB signaling, and it was analyzed whether a concerted action against ERa and ErbB signaling results in delay or prevention of treatment resistance.

Materials and methods

Inhibitors

Fulvestrant (ICI 182,780, Faslodex TM ) was obtained from Tocris (Avonmouth, Bristol, UK). The HER tyrosine kinase inhibitor CI-1033 (Canertinib) was donated by Pfizer Inc. (Groton, CT, USA), while SH-6 (Akt inhibitor) and RO-32-0432 (PKC inhibitor) were purchased from Calbiochem (Gibbstown, NJ, USA). The monoclonal antibody cetuximab (Erbitux) targeting EGFR was purchased from Merck (Glostrup, Denmark). Two ErbB2 targeting antibodies were used. Pertuzumab was a gift from Genentec (San Francisco, CA, USA), and trastuzumab was purchased from Roche (Hvidovre, Denmark).

Cell lines and culture conditions

The MCF-7 cell line was originally obtained from the Human Cell Culture Bank, Mason Research Institute (Rockville, MD, USA) and adapted to grow in low serum concentration (1%) to reduce the estrogens supplied through the serum to a level resembling postmenopausal concentrations of circulating estradiol [START_REF] Lykkesfeldt | Effect of estrogen and antiestrogens on cell proliferation and synthesis of secreted proteins in the human breast cancer cell line MCF-7 and a tamoxifen resistant variant subline, AL-1[END_REF]. The cells were maintained in growth medium without phenol red (DMEM/F12 medium (Gibco, Invitrogen, CA, USA)), supplemented with 1% heat-inactivated fetal calf serum (FCS) (Life Technologies, Bethesda, MD), 6 ng/ml bovine insulin (Novo Nordic, Bagsvaerd, Denmark), and 2 mM glutamax (Gibco). The fulvestrant resistant cell lines MCF-7/164 R -5 (164 R -5), MCF-7/164 R -7 (164 R -7), and MCF-7/182 R -6 (182 R -6) were established as described earlier [START_REF] Lykkesfeldt | Human breast cancer cell lines resistant to pure anti-estrogens are sensitive to tamoxifen treatment[END_REF] and maintained in growth medium supplemented with 0.1 lM fulvestrant. For experiments, 2.5 9 10 5 U penicillin and 31.25 lg/l streptomycin (Gibco) were added to the growth medium.

Cell proliferation assays

All cell lines were seeded in 24-well plates (2 cm 2 wells) in growth medium and allowed to adhere for 1-2 days. At the onset of experiment (day 0), growth medium containing inhibitors was added as indicated in the figure legends. Untreated controls were added similar amount of vehicle (ethanol or DMSO) as the treated cultures. Growth medium was replaced on day 3, and cell number determined on day 5 as previously described using a crystal violet colorimetric assay [START_REF] Lundholt | Growth inhibition and growth stimulation by estradiol of estrogen receptor transfected human breast epithelial cell lines involve different pathways[END_REF]. The obtained optical density (OD) for each sample was expressed as a relative value in percent of untreated control. Each experiment was performed in triplicate. For resistance development, MCF-7 cells were seeded in 24-well plates. The cells were allowed to attach for 24 h before treatment was initiated (day 0). Growth medium was changed twice every week and cell number was estimated with the colorimetric assay during the weeks 1, 2, 3, 4, 5, and 6 from onset of experiment. For these experiments, the actual OD was used as a measure for the cell number to the given time interval.

Western analysis

All cell lines were seeded in 6-well plates and cultured for 4-5 days in growth medium to obtain 70-80% confluent cultures. Cells were treated for 4 or 48 h with inhibitors as indicated in the figure legends and whole cell extracts were generated by lysing the cells in RIPA buffer (100 mM NaCl, 20 mM Tris-HCl, 1% Triton X-100, 0.5% sodiumdeoxycholate, 0.1% SDS and 1 mM EDTA, with the addition of 1 mM DTT, 1 mM NaF, 10 mM b-glycerolphosphate, 100 lM Na 3 VO 4 , 150 lM PMSF, and one tablet/10 ml complete mini protease inhibitor cocktail (Roche)). Protein concentration was determined using the BioRad Protein Assay kit (Bio-Rad Laboratories, Mu ¨nich, Germany). The proteins (10-15 lg per lane) were separated by electrophoresis on 4-12% Bis-Tris gels or 3-8% Tris Acetate gels (NuPage, Invitrogen) and transferred onto methanol-activated PVDF membranes (Immobilon-P, Millipore, Bedford, MA, USA). Non-specific binding of antibodies was prevented by incubating the membranes in TBS containing 5% dry-milk, 0.2% FCS, and 0.1% Tween-20 for 2 h at room temperature (RT). Incubation with primary antibodies was performed 1 h at RT or over night at 4°C depending on the antibody and followed by 1 h incubation with species-specific peroxidase-conjugated secondary antibodies (Dako, Glostrup, Denmark). Specific binding was detected by ECL PLUS (GE Healthcare, Hillerød, Denmark) and visualized using a CCD camera (LAS-1000, Fujifilm, Stockholm, Sweden). Quantification of protein expression levels was performed using the software Multi Gauge V2.2 (Fujifilm). In order to detect multiple proteins, the antibodies were removed from the membrane by incubation in 62.5 mM Tris-HCl, 100 mM b-mercaptoethanol and 2% (w/v) SDS, pH 6.7, and washed before incubation with antibody. The expression level of HSP70 (1:500.000, MS-482, Thermo Fisher Scientific, Fremont, CA, USA) was used as a loading control. The following antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): pAkt (1:500, 9271), Akt (1:2000, 9272), pErk1/2 (1:1000, 4377), and Erk1/2 (1:2000, 9102). The ERa (1:4000, RM-9101) antibody was purchased from Thermo Fisher Scientific. Bcl-2 antibody (1:2000, M0887) was purchased from Dako, Glostrup, Denmark.

Statistics

For all cell growth assays, three independent experiments were performed with triplicate or quadruplicate measures and the data were pooled for statistical analysis. Assumption of normality and homogeneity of variance was performed by graphical inspection of residuals. A mixed model of analysis of variance, followed by pair wise comparison with Bonferroni's correction was used to analyze for differences in response to treatment within and between cell lines. The given treatment and cell line were regarded as fixed variables, whereas the individual experiments were regarded as a random variable in the model. For experiments with combination therapy, data were log transformed in order to analyze data on a multiplicative scale, allowing for evaluation of interactions between the applied inhibitors. For experiments with resistance development in MCF-7 cells, each single experiment with triplicate measures was analyzed instead of pooling data from the three individual experiments. A linear model was used, followed by pair wise comparison with Bonferroni's correction. For all experiments, results were considered significant when P \ 0.05. Calculations were performed using SAS, version 9.1 (SAS institute, Cary, NC, USA).

Results

Cetuximab, but not trastuzumab or pertuzumab, inhibits growth of fulvestrant resistant cells

A prominent change in the fulvestrant resistant cell lines compared to parental MCF-7 cells is a significant upregulation of EGFR expression [START_REF] Frogne | Activation of ErbB3, EGFR and Erk is essential for growth of human breast cancer cell lines with acquired resistance to fulvestrant[END_REF]. Accordingly, we examined the effect of the antibody cetuximab on growth of parental MCF-7 cells and three fulvestrant resistant cell lines. As expected, treatment with cetuximab had no effect on the MCF-7 cell line that expresses a very low level of EGFR protein. For the resistant cell lines, only a modest growth inhibition (10%) was observed when 164 R -5 cells were treated with 50 lg/ml cetuximab (P = 0.02) and a 16 and 18% statistically significant growth inhibition was seen when 182 R -6 cells were treated with 5 lg/ml (P = 0.02) or 50 lg/ml (P = 0.01) cetuximab, respectively, Fig. 1a. No statistically significant growth inhibition was observed for 164 R -7 cells. ErbB2 is the preferred dimerization partner for the other ErbB receptors and especially the ErbB2/ ErbB3 dimer has been proposed to function as an oncogenic unit [START_REF] Holbro | The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation[END_REF]. Thus, we investigated the effect of ErbB2 targeting antibodies on cell growth to clarify if ErbB2 could play a role in antiestrogen resistant cell growth as a dimerization partner for ErbB3 as well as EGFR. However, there was not a significant effect on growth of MCF-7 cells or the resistant cell lines 164 R -5 or 164 R -7 when they were treated with the ErbB2 targeting antibodies trastuzumab (20 lg/ml) or pertuzumab (20 lg/ml), Fig. 1b.

CI-1033 preferentially inhibits growth of fulvestrant resistant cells and downregulates Erk signaling

In contrast to the limited effect of the monoclonal antibodies targeting EGFR or ErbB2, treatment with the new pan-ErbB inhibitor CI-1033 resulted in potent growth inhibition of all resistant cell lines, Fig. 2a. A preferential dose related growth inhibitory effect of the resistant cell Fig. 1 a Effect of the EGFR directed antibody cetuximab on cell growth of MCF-7 and the fulvestrant resistant cell lines 164 R -5, 164 R -7, and 182 R -6. Cells were treated for 5 days with the indicated concentrations of cetuximab before cell number was estimated by a colorimetric assay and expressed as percent of untreated control. Values (mean ± SD) are pooled data from three independent experiments, each performed with triplicate measures. * indicates values significantly different from the untreated control of the respective cell line. b Effect of the ErbB2 directed antibodies trastuzumab and pertuzumab on cell growth of MCF-7 and the fulvestrant resistant cell lines, 164 R -5 and 164 R -7. Cells were treated for 5 days with trastuzumab (20 lg/ml) or pertuzumab (20 lg/ml) before cell number was estimated by a colorimetric assay and expressed as percent of the untreated control. Values (mean ± SD) are pooled data from three independent experiments, each performed with triplicate measures lines compared to the unaffected parental MCF-7 cells was observed when treated with 0.02 lM (164 R -5 P = 0.005, 164 R -7 P = 0.007, and 182 R -6 P = 0.03), 0.1 lM (164 R -5 P \ 0.0001, 164 R -7 P \ 0.0001, and 182 R -6 P = 0.01), or 0.5 lM (164 R -5 P \ 0.0001, 164 R -7 P \ 0.0001, and 182 R -6 P \ 0.0001) CI. Thus, fulvestrant resistant cell lines were more dependent on ErbB signaling than the antiestrogen sensitive MCF-7 cells. 2 lM CI-1033 inhibited growth of MCF-7 cells to the same extent as the resistant cell lines, indicating general toxicity of the compound at this concentration. Both the PI3-K/Akt and the MEK/Erk pathway are commonly accepted to be downstream of ErbB signaling [START_REF] Holbro | The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation[END_REF] and we have previously shown upregulation of phosphorylated Akt (pAkt) and Erk (pErk) kinases in a panel of our resistant cell lines [START_REF] Frogne | Antiestrogen-resistant human breast cancer cells require activated protein kinase B/Akt for growth[END_REF][START_REF] Frogne | Activation of ErbB3, EGFR and Erk is essential for growth of human breast cancer cell lines with acquired resistance to fulvestrant[END_REF]. In the present study, a marked upregulation of pErk was found in the 164 R -5, 164 R -7, and 182 R -6 cell lines and a modest upregulation of pAkt was observed in the 164 R -5 and 164 R -7 cell lines, Fig 3a . The expression of pErk in the resistant cell lines was quantified by expressing the ratio pErk/Erk in percent of the ratio for MCF-7 cells. The expression of pAkt was not quantified due to the inaccuracy obtained when relating to a very low expressing control. Treatment of cells with CI-1033 effectively downregulated pErk expression in MCF-7 cells and all three resistant cell lines at concentrations from 0.1 lM, Fig. 3b. In order to quantify the observed changes in pErk expression, the pErk/Erk was determined for each treatment and expressed as a percent of the untreated control (Fig. 3b). CI-1033 treatment had no or only minor effect on pAkt expression (Fig. 3b). Again, the expression of pAkt was not quantified due to the inaccuracy obtained when relating to a low expressing control.

Combination therapy with CI-1033, SH-6, and RO-32-0432 is superior to single agent therapy

We have previously shown that growth of fulvestrant resistant cell lines also depend on Akt signaling and that treatment with the Akt inhibitor SH-6 has a preferential inhibitory effect on resistant cell growth [START_REF] Frogne | Antiestrogen-resistant human breast cancer cells require activated protein kinase B/Akt for growth[END_REF]. As Akt signaling appeared quite persistent despite CI-1033 treatment, we tested the effect of combined CI-1033 and SH-6 treatment on cell growth. PKCa also plays a role for growth of the resistant cell lines and the PKC inhibitor RO-32-0432 shows a preferential inhibitory effect on growth of resistant cells [START_REF] Frankel | Protein kinase C alpha is a marker for antiestrogen resistance and is involved in the growth of tamoxifen resistant human breast cancer cells[END_REF] independent of Erk or Akt signaling (Kristina Emdal, unpublished data). Therefore, we also tested the effect of a combination of CI-1033 and RO-32-0432 on growth of the cell lines. A low concentration of CI-1033 (0.1 lM), was used in combination with increasing concentration of SH-6 or RO-32-0432. In MCF-7 cells, 0.1 lM CI-1033 had no significant effect on cell growth alone and combination with SH-6 or RO-32-0432 was not superior to treatment with SH-6 or RO-32-0432 alone, Fig. 4a andb. In contrast, combination of CI-1033 and SH-6 was superior to treatment with each single agent in all the resistant cell lines, Fig 4a . For 164 R -5 the combination was superior at SH-6 concentrations of 1.5 lM (P = 0.02) and 2 lM (P = 0.002). For 164 R -7, the combination was superior at SH-6 concentrations of 1 lM (P = 0.03) and 1.5 lM (P = 0.03) and for 182 R -6 superior effects were seen at SH-6 concentrations of 1 and 1.5 lM (P \ 0.0001) and 2 lM (P = 0.02). For the combination of CI-1033 and RO-32-0432, a superior effect compared to single agent treatment was observed in all resistant cell lines when CI-1033 was combined with 2 lM RO-32-0432 (164 R -5 P = 0.008, 164 R -7 P = 0.02, and 182 R -6 P = 0.04), and 3 lM RO-32-0432 (164 R -5 P = 0.002, 164 R -7 P = 0.01, and 182 R -6 P \ 0.0001).

The growth inhibitory effect of CI-1033 is more pronounced in resistant cells grown in presence of fulvestrant

In order to investigate whether the effect of CI-1033 on the resistant cell lines was affected by the presence of fulvestrant, withdrawal experiments were performed as resistant cells are routinely cultured with fulvestrant in the medium. When fulvestrant was withdrawn from the resistant cell lines for one week prior to initiation of experiments, ERa expression was increased in all three resistant cell lines (compare fulvestrant cultures without CI-1033 and fulvestrant withdrawn cultures without CI-1033, Fig. 5). If ERa is functional, increased expression of ERa-regulated genes would be expected to follow fulvestrant withdrawal. Expression of the progesterone receptor (PR) is often used as an indicator of active ERa signaling. However, as fulvestrant resistant cell lines express extremely low PR levels and PR is not induced by estradiol in the resistant cells withdrawn from fulvestrant [START_REF] Larsen | Resistance of human breast-cancer cells to the pure steroidal antiestrogen ICI 182, 780 is not associated with a general loss of estrogen-receptor expression or lack of estrogen responsiveness[END_REF], the estrogen inducible Bcl-2 protein was selected as an indicator of functional ERa. A substantial increase in expression of Bcl-2 upon fulvestrant withdrawal was found in 164 R -5 and 164 R -7, but not in 182 R -6 (compare fulvestrant cultures without CI-1033 and fulvestrant withdrawn cultures without CI-1033, Fig. 5). Other studies have shown that a blockade of ErbB signaling could re-establish ERa expression and function [START_REF] Bayliss | Reversal of the estrogen receptor negative phenotype in breast cancer and restoration of antiestrogen response[END_REF][START_REF] Munzone | Reverting estrogen-receptor-negative phenotype in HER-2-overexpressing advanced breast cancer patients exposed to trastuzumab plus chemotherapy[END_REF]. In the present study, such an effect of ErbB blockade with CI-1033 was mainly expected in the fulvestrant withdrawn cultures, as the ERa protein is destabilized in the presence of fulvestrant. However, we did not observe any increase in ERa or Bcl-2 when fulvestrant witdrawn cultures were treated with CI-1033. In contrast, a slight increase in ERa expression was observed in the 182 R -6 fulvestrant cultures when treated with CI-1033, but the increase was minimal compared to the large increase observed upon fulvestrant withdrawal, indicating that ERa signaling in our resistant cell lines is mainly regulated by presence or absence of fulvestrant. Still, CI-1033 had a more pronounced growth inhibitory effect on all three resistant cell lines when added in combination with 0.1 lM fulvestrant at concentrations of 0.1 lM (164 R -5 P = 0.02, 164 R -7 P = 0.02, and 182 R -6 P = 0.04), 0.5 lM (164 R -5 P = 0.002, 164 R -7 P = 0.01, and 182 R -6 P = 0.005), or 1 lM (164 R -5 P = 0.02, 164 R -7 P = 0.01, 182 R -6 P \ 0.0001), Fig. 6. Thus, all resistant cell lines were able to partly escape the effects of CI-1033 treatment when released from fulvestrant-mediated blockade of the ERa receptor.

Heregulin-1b rescues MCF-7 cells from the growth inhibitory effects of fulvestrant

The lack of growth inhibition of MCF-7 cells with CI-1033 (see Fig. 2a) indicates that MCF-7 cells are independent of ErbB signaling when grown at standard conditions. Treatment of MCF-7 cells with fulvestrant significantly inhibits cell growth in a dose dependent manner, an effect that could partly be abrogated by co-treatment with 10 ng/ml of 1033 on total and phosphorylated forms of Akt and Erk in MCF-7 and the fulvestrant resistant cell lines 164 R -5, 164 R -7, and 182 R -6. HSP70 was used as loading control. Cells were treated for four hours with CI-1033 and lysed with RIPA buffer. Three independent experiments were performed and one representative blot is shown. To quantify the effect of CI-1033 on pErk expression, we expressed the ratio pErk/ Erk as a percentage of the ratio in the untreated control for each cell line the ErbB3/ErbB4 ligand heregulin-1b (Hrg) at all fulvestrant concentrations tested (P \ 0.0001), Fig. 7a. Thus, as also shown previously [START_REF] Larsen | Acquired antiestrogen resistance in MCF-7 human breast cancer sublines is not accomplished by altered expression of receptors in the ErbB-family[END_REF], activation of ErbB signaling can potently substitute for the fulvestrant-mediated blockade of ERa signaling in MCF-7 cells. When fulvestrant resistant cells are developed from MCF-7 cells by prolonged exposure to 0.1 lM fulvestrant, the cells show an initial arrest/death phase, followed by outgrowth of a small number of proliferating cell colonies after approximately 1 month [START_REF] Lykkesfeldt | Human breast cancer cell lines resistant to pure anti-estrogens are sensitive to tamoxifen treatment[END_REF]. Based on the finding that ErbB activation through Hrg treatment could rescue the effects of fulvestrant, we speculated whether a Hrg-mediated activation of the ErbB system could accelerate development of fulvestrant resistance. Therefore, MCF-7 cells were cultured for prolonged time (6 weeks) with 0.1 lM fulvestrant with or without 10 ng/ml Hrg. The cells treated with fulvestrant and Hrg displayed exponential growth and the cultures could be split every 10-12 days with a split ratio of about 10. Thus, switching on ErbB3/4 signaling by Hrg abolishes the growth inhibiting effects of even prolonged treatment with fulvestrant. However, long-term growth in presence of Hrg and fulvestrant (6 weeks) did not result in permanent fulvestrant resistance as withdrawal of Hrg resulted in cessation of growth, Fig. 7b. In order to examine how Hrg rescued the cells from the inhibitory effect of fulvestrant, different inhibitors were added in combination with fulvestrant and Hrg. Exposure to 50 lg/ml cetuximab had no effect on the Hrg-mediated rescue, whereas a partial inhibition of the Hrg-mediated rescue was observed when the long-term fulvestrant and Hrg cultures were treated with 20 lg/ml pertuzumab (P \ 0.0001) or 20 lg/ml trastuzumab (P \ 0.0001). A combination of 20 lg/ml trastuzumab and 20 lg/ml pertuzumab increased the inhibitory effect compared to single treatment with pertuzumab (P = 0.006) and this combination as well as treatment with CI-1033 alone, completely blocked the rescuing effect of Hrg (evaluated as not significantly different from the fulvestrant-only treated culture). Thus, the rescuing effect of Hrg was mediated through ErbB activation, likely through ErbB3 and/or ErbB4 dimerization with ErbB2. [START_REF] Massarweh | Resistance to endocrine therapy in breast cancer: exploiting estrogen receptor/growth factor signaling crosstalk[END_REF][START_REF] Gee | The antiepidermal growth factor receptor agent gefitinib (ZD1839/Iressa) improves antihormone response and prevents development of resistance in breast cancer in vitro[END_REF][START_REF] Okubo | Additive antitumour effect of the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib (Iressa, ZD1839) and the antioestrogen fulvestrant (Faslodex, ICI 182, 780) in breast cancer cells[END_REF]. Our findings that Hrg is able to restore growth of fulvestrant-treated MCF-7 cells and that MCF-7 sublines with acquired fulvestrant resistance are more sensitive to pan-ErbB inhibition when maintained on fulvestrant suggest that combined treatment targeting ErbB and ERa signaling could be used to prevent or delay development of treatment resistance. Prolonged exposure to 0.1 lM fulvestrant, 1-2.5 lM CI-1033, or a combination of 0.1 lM fulvestrant with 0.5-2 lM CI-1033 was performed in MCF-7 cells. Figure 8a shows the results from treatments with fulvestrant alone, 1 lM CI-1033 alone, and 0.5 lM CI-1033 in combination with fulvestrant. The MCF-7 culture treated with fulvestrant alone displayed a small initial increase in cell number followed by cell arrest and cell death. However, after 6 weeks a minor increase in cell number was observed, Fig. 8a. This increase was due to a few colonies of proliferating cells as shown in Fig. 8b. In the presence of 1 lM CI-1033, MCF-7 cells grew until confluence, yet slower than untreated cells, where after a balance between cell growth and cell death resulted in a steady state with a near-confluent cell density during the weeks 3-6, Fig. 8a. A comparable pattern was observed for cultures treated with 1.5 and 2 lM CI-1033 resulting in a stable population of viable cells after 6 weeks of treatment, Fig. 8b. MCF-7 cells could not sustain growth in presence of 2.5 lM CI-1033 for more than 2-3 weeks. It should be mentioned that MCF-7 cells could be subcultivated continuously with 1 lM CI-1033 with a weekly split ratio of between 10 and 15, compared to a split ratio for untreated MCF-7 cells of around 15-20. Cultures treated with a combination of fulvestrant and 0.5 lM CI-1033 only showed a small increase in cell growth over the first week, followed by cell arrest and cell death, Fig. 8a. Around week 5-6, small distinct colonies of cells appeared, Fig. 8b, but the cell number did not increase significantly above the cell number for day 0 during the course of this experiment, Fig. 8a. No colonies were visible in the cultures treated with 1 or 2 lM CI-1033 in combination with fulvestrant, Fig. 8b. Thus, long-term fulvestrant treatment results in a clonal selection of resistant cells, which could be prevented or delayed by a combination treatment strategy with CI-1033.

Discussion

Data from preclinical breast cancer models indicate that signaling through the ErbB tyrosine kinase receptors can promote antiestrogen failure in ERa positive breast cancer cells, e.g., through ectopic overexpression of TGFa, heregulin-1b, EGFR, and ErbB2 [START_REF] Arteaga | Blockade of the epidermal growth factor receptor inhibits transforming growth factor alpha-induced but not estrogen-induced growth of hormone-dependent human breast cancer[END_REF][START_REF] Pietras | HER-2 tyrosine kinase pathway targets estrogen receptor and promotes hormone-independent growth in human breast cancer cells[END_REF][START_REF] Van Agthoven | Ectopic expression of epidermal growth factor receptors induces hormone independence in ZR-75-1 human breast cancer cells[END_REF][START_REF] Benz | Estrogen-dependent, tamoxifen-resistant tumorigenic growth of MCF-7 cells transfected with HER2/neu[END_REF]. In concert, development of acquired resistance to antiestrogen therapy is often accompanied with changes resulting in increased signaling through the ErbB system, e.g., increased expression of ligands, receptors, or downstream effector molecules [START_REF] Mcclelland | Enhanced epidermal growth factor receptor signaling in MCF7 breast cancer cells after long-term culture in the presence of the pure antiestrogen ICI 182, 780 (Faslodex)[END_REF][START_REF] Atlas | Heregulin is sufficient for the promotion of tumorigenicity and metastasis of breast cancer cells in vivo[END_REF][START_REF] Sommer | Studies on the development of resistance to the pure antiestrogen faslodex in three human breast cancer cell lines[END_REF][START_REF] Frogne | Antiestrogen-resistant human breast cancer cells require activated protein kinase B/Akt for growth[END_REF][START_REF] Massarweh | Mechanisms of tumor regression and resistance to estrogen deprivation and fulvestrant in a model of estrogen receptor-positive, HER-2/neu-positive breast cancer[END_REF][START_REF] Fan | Diverse gene expression and DNA methylation profiles correlate with differential adaptation of breast cancer cells to the antiestrogens tamoxifen and fulvestrant[END_REF][START_REF] Frogne | Activation of ErbB3, EGFR and Erk is essential for growth of human breast cancer cell lines with acquired resistance to fulvestrant[END_REF][START_REF] Pancholi | ERBB2 influences the subcellular localization of the estrogen receptor in tamoxifenresistant MCF-7 cells leading to the activation of AKT and RPS6KA2[END_REF]. In the clinic, response to endocrine therapy is found to be reduced in patients with high expression of TGFa or EGFR [START_REF] Nicholson | Transforming growth factor-alpha and endocrine sensitivity in breast cancer[END_REF][START_REF] Nicholson | Epidermal growth factor receptor expression in breast cancer: association with response to endocrine therapy[END_REF], and ERa/ErbB2 positive tumors are less sensitive to both antiestrogen and aromatase inhibitor therapy than ERa positive/ErbB2 negative tumors [START_REF] Rasmussen | Adjuvant letrozole versus tamoxifen according to centrally-assessed ERBB2 status for postmenopausal women with endocrine-responsive early breast cancer: supplementary results from the BIG 1-98 randomised trial[END_REF]. In our cell culture model system comprising MCF-7 breast cancer cell lines with acquired resistance toward treatment with the antiestrogen fulvestrant, changes in the ErbB signaling pathway were evident at ligand, receptor, and downstream effector levels [START_REF] Frogne | Antiestrogen-resistant human breast cancer cells require activated protein kinase B/Akt for growth[END_REF][START_REF] Frogne | Activation of ErbB3, EGFR and Erk is essential for growth of human breast cancer cell lines with acquired resistance to fulvestrant[END_REF]. The present study examined the effects of ErbB inhibition in these fulvestrant resistant cell lines, alone and in combination with other signal transduction inhibitors as well as the effect of combined ErbB and ERa inhibition. The data showed that targeting either EGFR or ErbB2 with the monoclonal antibodies, cetuximab, pertuzumab, or trastuzumab, had limited or no effect on cell growth of parental MCF-7 cells and fulvestrant resistant sublines. In contrast, low concentrations of the pan-ErbB inhibitor CI-1033 showed preferential inhibition of the fulvestrant resistant cell lines ranging from 30 to 80% inhibition without affecting growth of MCF-7 cells. The superior effect of the tyrosine kinase inhibitor CI-1033 compared to antibodybased ErbB inhibition may likely be due to its pan-ErbB inhibitory effects. We have previously shown preferential growth inhibition of the resistant cell lines with 1 lM of the tyrosine kinase inhibitor gefitinib, a concentration believed only to inhibit the kinase activity of EGFR [START_REF] Frogne | Activation of ErbB3, EGFR and Erk is essential for growth of human breast cancer cell lines with acquired resistance to fulvestrant[END_REF]. This could appear to be in conflict with the modest/lacking effect of cetuximab observed in the present study, however in our cell lines 1 lM gefitinib did not only inhibit EGFR phosphorylation but also ErbB3 and ErbB4 phosphorylation ( [START_REF] Frogne | Activation of ErbB3, EGFR and Erk is essential for growth of human breast cancer cell lines with acquired resistance to fulvestrant[END_REF] and unpublished data for ErbB4), reinforcing the need of pan-ErbB inhibition for repression of resistant cell growth. It is becoming increasingly clear that the ErbB system of receptors is tightly coupled, pointing to the importance of dealing with these receptors as a complex network, which should be targeted in combination, rather than as individual receptors [START_REF] Citri | EGF-ERBB signalling: towards the systems level[END_REF]. A superior effect of targeting more than one ErbB receptor has also been shown in a breast cancer xenograft model, where a combination of pertuzumab, trastuzumab, and gefitinib more efficiently blocked tumor growth than either single agent [START_REF] Arpino | Treatment of human epidermal growth factor receptor 2-overexpressing breast cancer xenografts with multiagent HER-targeted therapy[END_REF]. Our resistant cell lines show increased expression of the ErbB3/ 4 ligand heregulin-2b and increased activity of ErbB3 [START_REF] Frogne | Activation of ErbB3, EGFR and Erk is essential for growth of human breast cancer cell lines with acquired resistance to fulvestrant[END_REF] and this increased ErbB3 signaling may in part explain the insensitivity to EGFR/ErbB2 antibody-based treatment as has previously been indicated in both clinical and preclinical studies [START_REF] Moulder | A phase I/II trial of trastuzumab and gefitinib in patients with metastatic breast cancer that overexpresses HER2/neu (ErbB-2)[END_REF][START_REF] Sergina | Escape from HER-family tyrosine kinase inhibitor therapy by the kinase-inactive HER3[END_REF]. However, it should be mentioned that the potent effects of CI-1033 compared to antibodybased therapy may not be solely ascribed to its pan-ErbB inhibitory effects as off-target inhibition of other tyrosine kinases cannot be ruled out. Early studies reported a high specificity of CI-1033 for the ErbB receptors [START_REF] Allen | Potential benefits of the irreversible pan-erbB inhibitor, CI-1033, in the treatment of breast cancer[END_REF], but this specificity has later been questioned as CI-1033 has been shown to be somewhat promiscuous in its targets [START_REF] Fabian | A small molecule-kinase interaction map for clinical kinase inhibitors[END_REF]. Yet, at low concentrations (0.1-0.5 lM) CI-1033 may be rather specific in targeting the ErbB receptor system in the present model system, based on the preferential effect in cell lines depending on ErbB signaling from growth. Overall, the potent and preferential growth inhibitory effect of low CI-1033 concentrations on antiestrogen resistant breast cancer cells clearly support the testing of CI-1033 in metastatic antiestrogen resistant breast cancer and may even suggest clinical activity in a subset of antiestrogen resistant breast cancers that do not respond to EGFR/ErbB2 monotherapy.

Erk and Akt are well known downstream effectors of ErbB signaling [START_REF] Citri | EGF-ERBB signalling: towards the systems level[END_REF]. In our cell lines, inhibition of ErbB signaling with CI-1033 primarily downregulated pErk and had little effect on pAkt. This is in agreement with our previous study, in which gefitinib-mediated inhibition of ErbB signaling in antiestrogen resistant cell lines resulted in reduced Erk signaling, but had no effect on the increased Akt signaling in resistant cell lines [START_REF] Frogne | Activation of ErbB3, EGFR and Erk is essential for growth of human breast cancer cell lines with acquired resistance to fulvestrant[END_REF], suggesting that this Akt activation is independent of ErbB signaling. We have previously shown that the Akt inhibitor SH-6 preferentially inhibited growth of tamoxifen and fulvestrant resistant cell lines compared to parental MCF-7 cells [START_REF] Frogne | Antiestrogen-resistant human breast cancer cells require activated protein kinase B/Akt for growth[END_REF] and therefore reasoned that a combination of CI-1033 and SH-6 could augment the growth inhibitory effects on resistant cells. Further, we have found that PKCa and d are overexpressed in tamoxifen and fulvestrant resistant cell lines and that the PKC inhibitor RO-32-0432 displayed preferential growth inhibition of resistant cell lines compared to parental MCF-7 cells [START_REF] Frankel | Protein kinase C alpha is a marker for antiestrogen resistance and is involved in the growth of tamoxifen resistant human breast cancer cells[END_REF]. As the effect of RO-32-0432 is not mediated through modulation of either Erk or Akt (Kristina Emdal, unpublished data), we also tested whether a combination of CI-1033 and RO-32-0432 treatment could augment the growth inhibitory effects on resistant cell lines. For both the combination of CI-1033 with SH-6 and the combination with RO-32-0432, a small superior effect on inhibition of resistant cell growth was observed compared to each single agent treatment. Thus, the data indicate that concomitant inhibition of signaling through Erk and Akt as well as Erk and PKC was superior to inhibition of a single pathway. The significant growth inhibition of resistant cells was seen at concentrations which had only little effect on parental cells, indicating that toxic side effects may be reduced by combination therapy.

Noteworthy, the present results indicate that both antiestrogen resistant breast cancer and presumably also a subgroup of primary antiestrogen sensitive breast cancers could benefit from adjuvant antiestrogen treatment combined with pan-ErbB targeted treatment. This is based on the observation that both our parental antiestrogen sensitive cells and the fulvestrant resistant cell lines had the ability to switch between ERa-and ErbB-mediated signaling, depending on externally applied factors. Fulvestrant is a very potent antiestrogen that downregulates ERa protein expression and has no agonistic activity [START_REF] Osborne | Fulvestrant: an oestrogen receptor antagonist with a novel mechanism of action[END_REF][START_REF] Wakeling | A potent specific pure antiestrogen with clinical potential[END_REF]. Fulvestrant resistance may be mediated through complete ERa repression, sometimes recognized as a loss of ERa expression and ERa independent activation of growth factor activity [START_REF] Osborne | Fulvestrant: an oestrogen receptor antagonist with a novel mechanism of action[END_REF][START_REF] Nicholson | Growth factor signalling and resistance to selective oestrogen receptor modulators and pure anti-oestrogens: the use of anti-growth factor therapies to treat or delay endocrine resistance in breast cancer[END_REF]. Our resistant cell lines display increased dependence on the ErbB pathway as evident from the increased sensitivity to the pan-ErbB inhibitor CI-1033 in the present study and to the EGFR inhibitor gefitinib in a previous study [START_REF] Frogne | Activation of ErbB3, EGFR and Erk is essential for growth of human breast cancer cell lines with acquired resistance to fulvestrant[END_REF]. Yet, when fulvestrant was withdrawn from the resistant cells, ERa expression was restored and in two out of three cell lines, expression of the estrogen responsive protein Bcl-2 followed, demonstrating the presence of a functional ERa in the resistant cells, in agreement with previously published data [START_REF] Larsen | Resistance of human breast-cancer cells to the pure steroidal antiestrogen ICI 182, 780 is not associated with a general loss of estrogen-receptor expression or lack of estrogen responsiveness[END_REF]. Other studies have shown that ErbB signaling in itself can suppress ERa expression and signaling in breast cancer cells and that inhibition of such ErbB signaling can reestablish ERa mediated signaling [START_REF] Oh | Hyperactivation of MAPK induces loss of ERalpha expression in breast cancer cells[END_REF][START_REF] Bayliss | Reversal of the estrogen receptor negative phenotype in breast cancer and restoration of antiestrogen response[END_REF]. This was not the case in our model system where ERa signaling, as visualized by ERa and Bcl-2 expression, was repressed by the presence of fulvestrant in the medium, but unaffected by ErbB blockade with CI-1033 upon faslodex withdrawal. Thus, ErbB signaling in the resistant cell lines did not appear to block ERa expression in itself. Further, in our fulvestrant withdrawn cells, the growth inhibitory effect of CI-1033 was significantly reduced compared to fulvestrant-treated cells, suggesting that regained ERa signaling could at least partly compensate for the inhibition of ErbB signaling with CI-1033. An ability to switch between ERa and ErbB2 signaling has recently been observed in our tamoxifen resistant cell line MCF-7/TAM R -1 when grown in presence and absence of tamoxifen [START_REF] Pancholi | ERBB2 influences the subcellular localization of the estrogen receptor in tamoxifenresistant MCF-7 cells leading to the activation of AKT and RPS6KA2[END_REF]. This is in line with xenograft models of tamoxifen resistance [START_REF] Arpino | Treatment of human epidermal growth factor receptor 2-overexpressing breast cancer xenografts with multiagent HER-targeted therapy[END_REF] and letrozole resistance [START_REF] Sabnis | Trastuzumab reverses letrozole resistance and amplifies the sensitivity of breast cancer cells to estrogen[END_REF], where ErbB blockade restored the antagonistic properties of the endocrine therapy, resulting in better effect of continued endocrine therapy with ErbB blockade than of ErbB blockade alone [START_REF] Sabnis | Trastuzumab reverses letrozole resistance and amplifies the sensitivity of breast cancer cells to estrogen[END_REF]. Also, ErbB2 overexpressing breast cancer cells with acquired resistance to ErbB directed therapy with lapatinib has been shown to switch from ErbB to ERa signaling for cell survival and growth [START_REF] Xia | A model of acquired autoresistance to a potent ErbB2 tyrosine kinase inhibitor and a therapeutic strategy to prevent its onset in breast cancer[END_REF]. In conclusion, our fulvestrant resistant cell lines clearly possessed the potential to use both ErbB and ERa signaling for growth stimulation, switching to one or the other pathway according to the inhibitors applied. Taken to the clinic, this support combined use of ErbB inhibitors and antiestrogen upon relapse on antiestrogen therapy.

Parental MCF-7 cells also possessed the ability to switch between ERa and ErbB signaling, as it could be demonstrated that addition of the ErbB3/ErbB4 ligand heregulin-1b (Hrg) prevented fulvestrant-mediated growth inhibition. Hrg-mediated rescue of fulvestrant inhibition of MCF-7 cell growth likely occured through ErbB3 and/or ErbB4 dimerization with ErbB2 as both trastuzumab and pertuzumab suppressed this stimulation and combination of the antibodies completely abolished Hrg-mediated growth stimulation. Hrg-mediated growth stimulation was also completely blocked by treatment with the pan-ErbB inhibitor CI-1033. Furthermore, we found that when treating MCF-7 cells with a combination of fulvestrant and CI-1033, outgrowth of resistant cells was delayed or prevented in the experimental period of 6 weeks. These data indicate that upfront treatment of breast cancer patients with agents targeting both ERa and ErbB signaling may delay development of resistance. A recent phase II neoadjuvant study supports the idea of combining endocrine therapy and inhibition of growth factor pathways in early breast cancer [START_REF] Baselga | Phase II randomized study of neoadjuvant everolimus plus letrozole compared with placebo plus letrozole in patients with estrogen receptor-positive breast cancer[END_REF]. The study showed that the efficacy of the aromatase inhibitor letrozole was significantly enhanced with concomitant targeting of mTOR, a kinase in the PI3 K/Akt pathway downstream of the ErbB receptors.

In conclusion, this study demonstrated that the new pan-ErbB inhibitor CI-1033 is very effective in blocking growth of antiestrogen resistant breast cancer cell growth at concentrations that do not affect antiestrogen sensitive cells. Further, both antiestrogen sensitive and antiestrogen resistant breast cancer cells showed the ability to switch between ERa and ErbB signaling depending on the applied treatment. Accordingly, the efficacy of CI-1033 treatment was increased when maintaining antiestrogen therapy, even after development of antiestrogen resistance. For parental antiestrogen sensitive cells, combined therapy against ERa and ErbB signaling significantly postponed or prevented the appearance of resistant cells. Thus, the present study provides support for combination of signal transduction inhibitors with endocrine agents as upfront therapeutic approach in human breast cancer as well as in patients relapsing on endocrine therapy.

5 Fig. 2

 52 Fig.2Effect of the pan-ErbB inhibitor CI-1033 on cell growth of MCF-7 and the fulvestrant resistant cell lines 164 R -5, 164 R -7, and 182 R -6. Cells were treated for 5 days before cell number was estimated by a colorimetric assay and expressed as percent of the untreated control. Values (mean ± SD) are pooled data from three independent experiments, each performed with triplicate measures. * indicates significant differences between the MCF-7 culture and three fulvestrant resistant cell lines after treatment with the indicated concentrations of CI-1033

Fig. 3 a

 3 Fig. 3 a Western blots showing expression of the total and phosphorylated levels of the Akt and Erk kinases in MCF-7 and the fulvestrant resistant cell lines 164 R -5, 164 R -7, and 182 R -6. HSP70 was used as loading control. Cells were grown until approximately 70% confluence and lysed with RIPA buffer. Three independent experiments were performed and one representative blot is shown. The expression of pErk in the resistant cell lines was compared to the expression in MCF-7 by expressing the ratio pErk/Erk as a percentage of the ratio in MCF-7 cells. b Western blots showing the effect of CI-

Fig. 4 aFig. 5 Fig. 6 Fig. 7 a

 4567 Fig.4a Effect of the Akt inhibitor SH-6 alone or in combination with the pan-ErbB inhibitor CI-1033 (CI) on growth of MCF-7 and the fulvestrant resistant cell lines 164 R -5, 164 R -7, and 182 R -6. Cells were treated for 5 days with the indicated concentrations of the inhibitors, before cell number was estimated by a colorimetric assay and expressed as percent of the untreated control. Values (mean ± SD) are pooled data from three independent experiments, each performed with triplicate measures. * indicates superior effect of the combined treatment compared to treatment with each single agent. b Effect of the PKC inhibitor RO-32-0432 (RO) alone or in combination with the pan-ErbB inhibitor CI-1033 (CI) on cell growth of MCF-7 and the fulvestrant resistant cell lines 164 R -5, 164 R -7, and 182 R -6. Cells were treated for 5 days before cell number was estimated by a colorimetric assay and expressed as percent of the untreated control. Values (mean ± SD) are pooled data from three independent experiments, each performed with triplicate measures. * indicates superior effect of the combined treatment compared to treatment with each single agent

Fig. 8 a

 8 Fig. 8 a Effect of long-term treatment of MCF-7 cells with fulvestrant (Fulv) alone or in combination with CI-1033 (CI). The cells were plated in 24-well plates and treatment initiated after 24 h (day 0) with CI-1033 (1 lM) or fulvestrant (0.1 lM) alone or CI-1033 (0.5 lM) in combination with fulvestrant (0.1 lM). Treatments were performed in triplicate and the cell number was determined by a colorimetric assay at day 0 and after 1, 2, 3, 4, 5, and 6 weeks. Values are mean ± SD from one representative out of three independent experiments, each performed with triplicate measures. * indicates cell numbers higher than the cell number at day 0. b Effect of long-term treatment of MCF-7 cells with fulvestrant (Fulv) and CI-1033 (CI) as single agents and in combination. The cells were plated in 24-well plates and treatment initiated after 24 h with CI-1033 and fulvestrant alone or fulvestrant in combination with CI-1033. Fulvestrant was administered in a dose of 0.1 lM and CI-1033 in doses as indicated in the figure. Treatment was performed in triplicate and the cells were stained with crystal violet at day 0 and after 1, 2, 3, 4, 5, and 6 weeks. Picture from a representative culture plate stained at week 6 is shown

Breast Cancer Res Treat (2010) 121:601-613

Acknowledgment We gratefully acknowledge the excellent technical assistance from Inger Heiberg. This study was supported by grants from Danish Cancer Society, Danish Agency for Science Technology and Innovation 271-07-0409, and Beckett Foundation and Mrs. Astrid Thaysen's Foundation for Basic Medical Research.


P N Span

H W Van Laarhoven

F C G J Sweep

Á F C G J Sweep

Tamoxifen sensitivity and estrogen receptor mRNA levels

To the editor, In a recent letter, Smith et al. [START_REF] Smith | Estrogen receptor regulation: don't forget translation[END_REF] respond to an article from Pentheroudakis et al. [START_REF] Pentheroudakis | Gene expression of estrogen receptor, progesterone receptor and microtubule-associated protein Tau in high-risk early breast cancer: a quest for molecular predictors of treatment benefit in the context of a Hellenic Cooperative Oncology Group trial[END_REF] in Breast Cancer Research and Treatment concerning the use of the quantitative RT-PCR (qPCR) for Estrogen Receptor alpha (ER) as an alternative for the immunohistochemical (IHC) determination of ER status in breast cancer tissue. Smith et al. state that the lack of correlation between qPCR and IHC shown in this article [START_REF] Pentheroudakis | Gene expression of estrogen receptor, progesterone receptor and microtubule-associated protein Tau in high-risk early breast cancer: a quest for molecular predictors of treatment benefit in the context of a Hellenic Cooperative Oncology Group trial[END_REF] might be due to the various regulatory translation mechanisms that will cause differences in efficiency of translation from mRNA to protein. They conclude that because of this poor relation between mRNA and protein, it is unlikely that transcriptional profiling can be used for predicting benefit from hormonal therapy in breast cancer.

We would like to make a few remarks on this issue. Firstly, the correlation between a quantitative assay as qPCR and the semi-quantitative nature of IHC might be inherently low. The ligand binding assay (LBA) for measuring estradiol binding in tissue for which we have performed quality assurance [START_REF] Geurts-Moespot | Twenty years of experience with the steroid receptor external quality assessment program-the paradigm for tumour biomarker EQA studies. On behalf of the EROTC Receptor and Biomarker Study Group[END_REF] is much more quantitative than IHC. Indeed, in our patient cohort [START_REF] Span | Mammaglobin is associated with low-grade, steroid receptor-positive breast tumors from postmenopausal patients, and has independent prognostic value for relapsefree survival time[END_REF] (n = 243), we found a strong significant correlation (R s = 0.776, P \ 0.001) between the LBA assay and qPCR data for ER. At a similar cut-off point for both LBA and qPCR, we found a Kappa of 0.76, which denotes substantial agreement [START_REF] Pentheroudakis | Gene expression of estrogen receptor, progesterone receptor and microtubule-associated protein Tau in high-risk early breast cancer: a quest for molecular predictors of treatment benefit in the context of a Hellenic Cooperative Oncology Group trial[END_REF].

Importantly, the amount of ER mRNA was specifically predictive for disease-free survival in the Tamoxifen-treated patients in our cohort (n = 82), when entered as a continuous factor in Cox regression (hazard ratio (HR) = 0.508, 95% confidence interval (CI) = 0.348-0.747, P = 0.001). At the cut-off point mentioned above, we find an HR = 0.251 (CI = 0.127-0.497, P \ 0.001, Fig. 1).

Finally, we do not agree with the broad statement that ''this casts doubt on the use of transcriptional profiling for predicting pathway activities or benefit from hormonal therapy in breast cancer.'' We strongly believe that the rapidly expanding knowledge on how different ER ligands regulate gene expression [START_REF] Welboren | ChIP-Seq of ERalpha and RNA polymerase II defines genes differentially responding to ligands[END_REF][START_REF] Welboren | Genomic actions of estrogen receptor {alpha}: what are the targets and how are they regulated?[END_REF][START_REF] Fullwood | Fig. 1 Disease-free survival of patients treated with Tamoxifen, after dichotomization based on high (dashed line) or low (straight line) ER mRNA levels[END_REF] will soon allow us to quantitatively profile the expression levels of the target genes of the ER; thus, providing a sensitive and quantitative measure of estrogen sensitivity of the tumor.


Brigitte Se ´radour •

Hubert Allemand

Alain Weill

Philippe Ricordeau

Sustained lower rates of breast cancer incidence in France in 2007

We previously reported a decrease in breast cancer incidence between 2003 and 2006 for only women aged 50 or above in France [START_REF] Allemand | Decline in breast cancer incidence in 2005 and 2006 in France : a paradoxical trend[END_REF]. The magnitude and timing of decrease varied by age [START_REF] Se ´radour | Changes by age in breast cancer incidence, mammography screening and hormone therapy use in France from 2000 to[END_REF]. This was in the context of a substantial reduction in hormonal replacement therapy (HRT) prescriptions after 2001.

In this update we report that, this decrease has been maintained into 2007 only for the women 55-64 years old. We used the same national data sources on HRT prescribing and on breast cancer in France up to 2007 [START_REF] Se ´radour | Changes by age in breast cancer incidence, mammography screening and hormone therapy use in France from 2000 to[END_REF].

The number of HRT users has dropped by 64% between 2001 and 2007, and remained low after 2004 compared to 2001. The largest decrease was observed in women 55-64 years old, the group in which the prevalence of HRT use has been highest and the decrease most pronounced: 32% in 2001, 19% in 2004, and 11% in 2007. Recent publication from Sweden also reported a significant decrease in incidence confined to women 50-59 years of age, in the same context of dramatic decline in the use of HRT [START_REF] Lambe | Reductions in use of hormone replacement therapy: effects on B. Se ´radour (&) ARCADES (Association pour la recherche et le de ´pistage des cancers du sein, du col de l'ute ´rus et des cancers colorectaux)[END_REF].

Figure 1 shows that breast cancer incidence rates in France in women aged 55-59 and 60-64 years old decreased 14.7% [95% CI 12.2 to 17.2%] and 12.6% [95% CI 10.

  1 to 15.1%] from 2003 to 2007. No significant change was observed in the age groups 50-54 and 65-69. The slight decline observed in those age groups between 2003 and 2006 (0.7 and 2.1%, respectively) was followed by an increase in 2007. Mammography use increased continuously in France between 1990 and 2005, and was stable in 2006 and 2007 after the implementation of the National Breast Cancer Screening Programme in 2004. Participation rates for French women aged 50-74 years were 40.2% in 2004 increasing to 50.8% in 2007. The upward trend concerned all age groups. Digital mammography was allowed in the screening programme from 2008. Gradual development of screening and change in technology cannot account for the reduction in breast cancer incidence. Breast cancer trends in France are thus broadly similar to those of Western countries where prevalence of HRT use was high [4-9].Differences between countries may be the reflect of different types of HRT and duration of use.In France the range of HRT preparations differs from what is found in the United States, but our results indicate also in France a striking correlation between fall in HRT use and the decline in incidence of breast cancer, in women older than 55 years.

	600
	Peak of HRT use
	500
	400
	300
			50-54	55-59	60-64	65-69
	200
		Implementation of the French Breast
	100	Cancer Screening Programm
	0
	S2-1999 S1-2000 S2-2000 S1-2001 S2-2001 S1-2002 S2-2002 S1-2003 S2-2003 S1-2004 S2-2004 S1-2005 S2-2005 S1-2006 S2-2006 S1-2007 S2-2007
	Year (S1: January ; S2: June)
	Swedish breast cancer incidence trends only seen after several	7. Canfell K, Banks E, Clements M et al (2009) Sustained lower rates
	years. Breast Cancer Res Treat. doi: 10.1007/s10549-009-0615-7	of HRT prescribing and breast cancer incidence in Australia since
	4. Kumle M (2008) Declining breast cancer incidence and decreased	2003. Breast Cancer Res Treat. doi: 10.1007/s10549-009-0331-3
	HRT use. Lancet 372:608-610		8. Katalanic A, Rawal R (2008) Decline in breast cancer incidence
	5. Cronin KA, Ravdin PM, Edwards BK (2008) Sustained lower rates	after decrease in utilisation of hormone replacement therapy.
	of breast cancer in the United States. Breast Cancer Res Treat. doi:	Breast Cancer Res Treat 107:427-430
	10.1007/s10549-008-0226-8		9. Bouchardy C, Usel M, Verkooijen HM, Fioretta G et al (2009)
	6. Jemal A, Ward E, Thun MJ (2007) Recent trends in breast cancer	Changing pattern of age-specific breast cancer incidence in the
	incidence rates by age and tumor characteristics among U.S.	Swiss canton of Geneva. Breast Cancer Res Treat. doi: 10.1007/
	women. Breast Cancer Res 9:R28. doi:10.1186/bcr1672		s10549-009-0478-y

age Fig. 1 Breast cancer incidence by age in France


Luca Gallelli
email: gallelli@unicz.it

Manuela Colosimo

Grazia A Tolotta

Daniella Falcone

Laura Luberto

Lucia S Curto

Pierandrea Rende

Francesca Mazzei

Norma M Marigliano

Giovambattista De Sarro

Giovambattista De

Salvatore Cucchiara

Prospective randomized double-blind trial of racecadotril compared with loperamide in elderly people with gastroenteritis living in nursing homes

Keywords: Acute diarrhea, Gastroenteritis, Elderly patients, Racecadotril, Loperamide

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Gastroenteritis is an inflammation of the gastrointestinal tract involving both the stomach and the small intestine and resulting in acute diarrhea. Inflammation is caused most often by infection with certain viruses and less often by bacteria or their toxins, parasites, or adverse reaction to something in the diet or drugs [START_REF] Aranda-Michel | Acute diarrhea: a practical review[END_REF]. Whereas gastroenteritis normally has a mild clinical course, hospitalization and death are more common among elderly people, particularly those who live in geriatric nursing homes. In fact, gastroenteritis will occur in settings where people gather, even when standards of care and food hygiene are very high [START_REF] Frenzen | Mortality due to gastroenteritis of unknown etiology in the United States[END_REF]. Several factors, such as the physiological decrease in gastric acid secretion, are involved in the increased vulnerability to diarrhea in elderly people. Other factors, such as decreased motility of the gastrointestinal tract; reduced immune response; residence in nursing homes, retirement communities, and hospitals with crowded conditions; and a possible increased mucosal sensitivity to toxins; all carry greater risk to the elderly [START_REF] Hunter | Pharmacotherapeutics in older adults[END_REF][START_REF] Shaojun | The clinical implications of ageing for rational drug therapy[END_REF]. Some infections are inevitable due to the susceptibility of the population and the highly infectious and persistent nature of enteric pathogens. Several enteropathogenic viruses, i.e., caliciviruses, norovirus, and rotavirus, are the most common causal agents in acute gastroenteritis outbreaks [START_REF] Evans | General outbreak of infectious disease in England and Wales: 1995 and 1996[END_REF][START_REF] Nilsson | Incidence and genetic diversity of group c rotavirus among adults[END_REF].

Gastroenteritis usually appears with acute diarrhea, abdominal pain, and vomits. Acute diarrhea represents a common clinical problem in developed countries and epidemic plague in underdeveloped nations. It is calculated to be responsible worldwide for approximately 2 million deaths per year [START_REF] Kosek | The global burden of diarrhoeal disease, as estimated from studies published between 1992 and 2000[END_REF]. In the United States, approximately half those who die of diarrhea are of advanced age to 74 years, with increased total mortality about 400 times greater than other ages [START_REF] Bennett | Approach to acute diarrhea in the elderly[END_REF].

Managing an elderly patient with diarrhea must ensure proper hydration using available oral rehydration solutions (ORS) [START_REF] Slotwiner-Nie | Infectious diarrhea in the elderly[END_REF][START_REF] Trinh | Diarrheal diseases in the elderly[END_REF]. In many patients, however, this is not possible due to their decreased ability to drink. Therefore, other approaches may be used in reduce the risk of dehydration and systemic or metabolic diseases. Available drugs are represented by loperamide and racecadotril. Loperamide is an mu-opiate-receptor agonist, which prolongs orocecal and colonic transit times by disrupting the gut's electrical activity, increasing gut capacity, and delaying the passage of fluids through the small intestine [START_REF] Shahbazian | Involvement of mu-and kappa-, but not delta-, opioid receptors in the peristaltic motor depression caused by endogenous and exogenous opioids in the guinea-pig intestine[END_REF]. Racecadotril, an enkephalinase inhibitor, is an indirect delta receptor agonist, with antisecretory activity [START_REF] Huijghebaert | Racecadotril versus loperamide: antidiarrheal research revisited[END_REF][START_REF] Prado | Global Adult Racecadotril Study Group (2002) A multinational comparison of racecadotril and loperamide in the treatment of acute watery diarrhoea in adults[END_REF] without activity on gut muscle [START_REF] Bergmann | Effects of acetorphan, an antidiarrhoeal enkephalinase inhibitor, on oro-caecal and colonic transit times in healthy volunteers[END_REF].

The aim of this study was to compare the efficacy and tolerability of loperamide and racecadotril in elderly patients residing in a geriatric nursing home who had severe acute diarrhea. Moreover, to evaluate the role of metabolism in both efficacy and tolerability, we evaluated cytochrome P450 polymorphisms. Finally, at the end of the study, we performed a pharmacoeconomic evaluation of both treatment regimens.

Methods

The study was a randomized, prospective, double-blind, and parallel group design implemented geriatric nursing homes in Catanzaro, Italy, from February 2008 to March 2009. Eligible patients were women and men with acute diarrhea (defined as three or more liquid stools in 24 h), without signs of severe dehydration (sunken eyes, reduced skin turgor, lethargy, unable to drink, and absent/uncountable radial pulse) and bacterial infection (i.e., the presence of blood or pus in their stool). Patients were excluded if they met any of the following conditions: presence of chronic or iatrogenic diarrhea, temperature >38.6°C, signs or symptoms of orthostatic hypotension, inability to take medications and fluids by mouth; a history of chronic gastrointestinal tract disease, hepatic or renal insufficiency, or a history of hypersensitivity to loperamide or racecadotril. Finally, patients who had taken any antidiarrheal or antimicrobial drugs in the 5 days prior to the study, and patients with concomitant illnesses requiring antibiotic treatment, were not eligible.

Experimental protocol

Patients with acute diarrhea and without regard for age, sex, or medical history were randomly allocated to receive either one tablet of racecadotril 100 mg every 8 h or two tablets of loperamide 2.0 mg followed by one tablet after each unformed stool, up to four tablets in any 24-h period. For the randomization, was used a computer program to generate a sequence of treatment allocations by block randomization using a random number generator. Investigators were blinded to the block size to avoid selection bias. Each treatment was started after the third diarrhea episode and was stopped after the production of two consecutive normal stools or no stool production for a period of 12 h. In order to exclude gut infection, before treatment in both groups, a sample of fresh stool was collected for microbiological analysis. If recovery did not occur within 4 days, the treatment was discontinued and, in order to avoid dehydration, the patient was treated with other drugs. Each dose of the medication was taken under the supervision of a designated study physician. Time, number, and type of stool were recorded, as were the occurrence of adverse signs and symptoms. In both groups, 750 ml of a standard ORS [sodium (Na+) 90 mmol/l, chlorine (Cl2) 80 mmol/l, potassium (K+) 20 mmol/l, citrate 10 mmol/l, and glucose 111 mmol/l] was given to all patients every day. The study was approved by Ethics Committee and was conducted in accordance with the Declaration of Helsinki. Written informed consent was received from each patient. For patients with senile dementia, consent was obtained from a partner.

Microbiological analysis

Fresh stool samples were collected at the beginning of the study by the nursing staff and sent for routine microbiological analysis for Salmonella spp., Shigella spp., Yersinia spp., Vibrio spp., Campylobacter spp., Staphylococcus aureus and intestinal parasites (helminthes and protozoa), according to standard protocols.

Evaluations

The primary efficacy criterion was the duration of diarrhea in days, from the first treatment dose to recovery. Secondary efficacy criterion consisted of duration of other symptoms, i.e., abdominal pain and abdominal distension. The overall clinical response during the study was assessed by physicians who were blinded to the treatment. Stool output, electrolytes, fluid intake, body weight, and hematocrit were also evaluated weekly in both groups. Stool output was calculated as the sum of the weights of the watery and loose stools (diarrheic stools) divided by the body weight at base line. Tolerability and safety were assessed by recording the adverse events experienced during treatment and by the occurrence of constipation. Signs and symptoms evaluated were pain on abdominal palpation, anorexia, nausea, and anal burning. A visual analogue scale (VAS) was used to assess pain severity before, during, and after treatment. Pharmacoeconomic evaluation was performed at the end of study period, and both intent-to-treat (ITT) and per-protocol (PP) costs of drug treatments only were analyzed. We did not consider indirect costs, because patients were residing in nursing homes, with both physicians and nursing staff presents every day. We estimated the cost of the drugs using average wholesale prices. Racecadotril (20 tablets of 100 mg) was estimated at 11.50 euros (0.575 euros for each tablet), and the price of loperamide (15 tablets of 2 mg) was 5 euros (0.33 euros for each tablet). The defined daily dose, which represents the assumed average daily dose of a drug when used for its main indication in adults, was 1.725 euros for racecadotril and 1.65 euros for loperamide (a mean of five tablets/day). To evaluate the role of genetic polymorphisms in cytochrome P450 involved in drug metabolism, a blood sample was taken from each patient at the beginning and end of the study period. Finally, we analyzed polymorphisms in patients nonresponsive to treatment and in patients who developed side effects.

Statistical analysis

Statistical analysis was performed using SPSS. Both ITT and PP analyses were performed for primary and secondary parameters. Estimates of the survival distribution of the duration of diarrhea were analyzed using Kaplan-Meier survival analysis techniques. The overall clinical response of the treatment groups was compared using the Cochran-Mantel-Haenszel test, as was the comparison of treatment group changes from inclusion to the end of treatment.

Results

Patient characteristics

During the study period, 61 patients (19 men and 42 women) were enrolled for ITT and randomized in two groups; 30 (nine men and 21 women) were treated with racecadotril, whereas 31 patients (ten men and 21 women) received loperamide (data not shown). At the end of the study, 54 patients participated fully, making up the PP population (88.5%). Two patients (3.27%) did not complete the study due to adverse events, three 4.9 %) were considered noncompleters due to deviation from protocol, and two (3.27%) were lost to follow-up. Mean ages were 82.9 (range 73-96) years for the racecadotril group and 81.9 (range 73-93) years for the loperamide group (Fig. 1). Other variables with regard to body weight, duration of diarrhea, and fluid intake were comparable between the two groups. All patients presented polytherapy, but pharmacological evaluation performed by a specialist excluded that drug treatments may be involved in the development of diarrhea. Microbiological investigation performed on fresh stool taken before treatment documented that fecal specimens were negative for bacterial enteropathogens and intestinal parasites (data not shown).

Symptoms and stool normalization

During the study period, the racecadotril group experienced a mean of 3.93 episodes of acute diarrhea [range 1-11 episodes; standard deviation (SD) 2.99] after they had met the inclusion criteria (three episodes in 24 h), and a mean of 7.29 in the loperamide group (range 2-16 episodes; SD 4.45) (data not shown). We defined an episode as all diarrhea manifestations in each patient from the beginning of stool output alteration up to stool output normalization. Before the beginning of the treatment, in both groups, stool output comprised between 260 and 270 g/kg. In both group, all episodes of diarrhea were more common in women, with no difference in the median age (Fig. 1). The mean duration of abdominal pain in the ITT population was 14± 1 h for racecadotril and 28±1 h for loperamide. In the PP population, the mean duration of abdominal pain was 14± 1 h for racecadotril and 32±2 h for loperamide (P<0.01). In all episodes, normal stools were collected 36±4 h after the beginning of racecadotril and 63 ±6 h from the beginning of loperamide (P <0.01) (data not shown). Mean total stool output before recovery was 120±27 g kg in the racecadotril group and 150±39 g/kg in the loperamide group, a 25 percent lower output with racecadotril (P<0.01). In about 50% of patients (age 80± 0.2 years; range 73-93 years), in the loperamide group during the study period, 4 days of treatment were ineffective. Therefore, patients were treated, after a new negative microbiological evaluation, with racecadotril, with a rapid normalization of all symptoms. There were no statistically significant differences between the two treatment groups for the major outcome variables: ORS intake and need for unscheduled intravenous fluids.

Adverse drug reactions

About 50% of patients experienced at least one adverse event during the study: 12% in the racecadotril group and 60% in the loperamide group. The most frequently occurring adverse events were nausea and constipation. In particular, nausea occurred in 10% of patients treated with racecadotril and 20% of patients treated with loperamide; constipation was observed in 15% of patients in the racecadotril group and in 60% in the loperamide group. Adverse events noted as per protocol, such as abdominal pain, headache, anorexia, etc., were not different between groups. During the study, at the end, and at follow-up, no electrolyte alterations were recorded in either group.

Genetic evaluation

To evaluate the role of cytochrome (CYP) metabolism in loperamide failure, blood of patients not responsive to loperamide was analyzed for the main allelic variants of the CYP450 isoforms 3A4/5 and CYP2C8. CYP2C8 and allelic variants of CYP3A4 genes were analyzed with real-time reverse-transcriptase polymerase chain reaction (PCR) by TaqMan kits purchased from Applied Biosystems. Patients did not carry any detrimental CYP2C8 or CYP3A4 allele. Furthermore, they did not carry extra copies of functional alleles CYP2C8 or CYP3A4; therefore, they were not genetically classifiable as ultrarapid or poor metabolizers for these two enzymes.

Pharmacoeconomic evaluation

Pharmacoeconomic analysis performed at the end of our study documented an increase in costs in the loperamide group with respect to racecadotril. Based on the ITT analysis, costs average in the racecadotril group was 44.85 euros and for the loperamide group 91.99 euros (Fig. 2).

Based on PP analysis, mean cost of treatment was 44.85 euros in the racecadotril group compared with 107.5 euros for the loperamide group (Fig. 2). Based on both ITT and PP analysis, loperamide treatment was associated with an incremental total cost (Fig. 2).

Discussion

In this study, we evaluated the effects of loperamide and racecadotril on acute diarrhea in elderly people living in nursing homes. There are multiple causes of gastroenteritis and acute diarrhea, including ingestion of toxins, food intolerance, psychological stress, and adverse reactions to medications. In most cases, the cause is thought to be Fig. 1 Mean patient age during all episodes of diarrhea in both racecadotril (black) and loperamide (white) treatment groups infections, including those caused by viruses, bacteria, or parasites [START_REF] Guerrant | Bacterial and protozoal gastroenteritis[END_REF][START_REF] Blacklow | Viral gastroenteritis[END_REF]. Viruses are more important in infants (rotaviruses) [START_REF] De Wit | Sensor, a population-based cohort study on gastroenteritis in the Netherlands: incidence and etiology[END_REF][START_REF] Dupont | What's new in enteric infectious diseases at home and abroad[END_REF][START_REF] Marsella | Epidemiology of rotavirus-associated hospital admissions in the province of Ferrara, Italy[END_REF] and cause community foodand water-borne gastroenteritis (noroviruses) in all age groups [START_REF] Chadwick | Transmission of a small round structured virus by vomiting during a hospital outbreak of gastroenteritis[END_REF][START_REF] Parashar | Norwalk-like viruses". Public health consequences and outbreak management[END_REF]. In health-care facilities, transmission can additionally occur through hand transfer of the virus to the oral mucosa via contact with materials, vomits, and environmental surfaces that have been contaminated with either feces or vomits. These circumstances make it extremely difficult to control outbreaks in institutional settings [START_REF] Said | Healthcare epidemiology: gastrointestinal flu: norovirus in health care and long-term care facilities[END_REF][START_REF] Cheesbrough | Widespread environmental contamination with Norwalklike viruses (NLV) detected in a prolonged hotel outbreak of gastroenteritis[END_REF].

In developing countries, bacterial enteropathogens, i.e., salmonella, shigella, Campylobacter jejuni,, enteropathogenic Escherichia coli, and vibrio cause just under half of all cases of endemic pediatric diarrhea and approximately half the cases of diarrhea among international travelers to these areas [START_REF] Jiang | Prevalence of enteric pathogens among international travelers with diarrhea acquired in Kenya (Mombasa), India (Goa), or Jamaica (Montego Bay)[END_REF]. In our we enrolled 61 elderly patients residing in a geriatric nursing home who had no signs and symptoms of acute bacterial gastroenteritis. To exclude further patients with bacterial diarrhea, a microbiological analysis of fresh stool was taken from each patient before treatment. Microbiological analysis excluded the presence of bacterial enteropathogens or intestinal parasites.

Usually, the pathophysiological problem of acute diarrhea due to nonbacterial agents is induced by hypersecretion of intestinal mucosa. Therefore, the ideal treatment for acute diarrhea should combine replacement ORS and drugs able to modulate intestinal hypersecretion or gastrointestinal transit, such as racecadotril and loperamide. Both racecadotril and loperamide are rapid, equally effective, treatments for acute diarrhea in adults [START_REF] Wang | A blind, randomized comparison of racecadotril and loperamide for stopping acute diarrhea in adults[END_REF], do not penetrate the nervous system, and have no substantial potential for addiction. However, they have several differences in pharmacodynamic and pharmacokinetic parameters. Racecadotril, a specific enkephalinase inhibitor, prevents inactivation of endogenous enkephalins and prolongs their physiologic action. Enkephalins act as neurotransmitters in the gastrointestinal tract by activating delta-opiate-receptors and thus reducing the level of cyclic adenosine monophosphate (AMP) [START_REF] Rachmilewitz | Effect of opiates on human colonic adenylate cyclase activity[END_REF]. The result is a low secretion of water and electrolytes without any detectable effect on intestinal motility [START_REF] Bergmann | Effects of acetorphan, an antidiarrhoeal enkephalinase inhibitor, on oro-caecal and colonic transit times in healthy volunteers[END_REF][START_REF] Shook | Antidiarrhoeal properties of supraspinal mu and delta and peripheral mu, delta and kappa opioid receptor: Inhibition of diarrhea without constipation[END_REF]. The efficacy and safety of racecadotril as an adjuvant to ORS therapy were documented several years ago in infants and children with less severe diarrhea [START_REF] Cézard | Efficacy and tolerance of acetorphan in infant acute diarrhea: a multicentric, double blind study[END_REF] and in children with rotavirusassociated diarrhea [START_REF] Salazar-Lindo | Racecadotril in the treatment of acute watery diarrhea in children[END_REF]. Hamza et al. [START_REF] Hamza | Racecadotril versus placebo in the treatment of acute diarrhoea in adults[END_REF] and Vetel et al. [START_REF] Vetel | Comparison of racecadotril and loperamide in adults with acute diarrhoea[END_REF] reported that racecadotril produced a significant (P=0.025) decrease in stool weight during the first day of treatment compared with placebo and was also associated with significantly fewer diarrheic stools than placebo after 1 day of treatment. Other studies in adult patients reported that racecadotril was able to reduce diarrhea in about 19 h [START_REF] Wang | A blind, randomized comparison of racecadotril and loperamide for stopping acute diarrhea in adults[END_REF][START_REF] Rouge | The enkephalinase inhibitor, acetorphan, in acute diarrhea: a double-blind control clinical trial versus loperamide[END_REF]. In agreement with these studies, we documented that racecadotril reduced diarrhea in all patients in about 1 day in all episodes of gastroenteritis. No ineffectiveness was recorded during this treatment. Loperamide is a selective mu-opiate-receptor agonist that is able to prolong orocecal and colonic transit times by disrupting the gut's electrical activity, increasing gut capacity, and delaying the passage of fluids through the small intestine [START_REF] Basilisco | Oral naloxone antagonizes loperamide-induced delay of orocecal transit[END_REF]. Several studies reported that loperamide is able to reduce diarrhea manifestations in adults, with or without antibiotic treatments [START_REF] Riddle | Effect of adjunctive loperamide in combination with antibiotics on treatment outcomes in traveler's diarrhea: a systematic review and meta-analysis[END_REF].

Of the 61 patients randomized in our study (ITT population), 54 (28 in the racecadotril group and 26 in the loperamide group) were considered valid as per protocol. Three patients with moderate Alzheimer's dementia ([Mini Mental State Examination (MMSE) score of 15] were excluded for the impossibility of evaluating abdominal pain intensity with the VAS scale and also evaluating the impact of side effects. We documented that loperamide reduced diarrhea in about 3 days in all episodes of gastroenteritis. However, in about 50% of treated patients over the entire treatment period, it was ineffective. The percentage of Fig. 2 Estimated cost for defined daily dose (DDD) of both racecadotril and loperamide treatments for each episodes of diarrhea calculated as intent to treat (ITT) (up) and per protocol (PP) (down). We define an episode of diarrhea as all diarrhea manifestations in each patient from stool output alteration to stool output normalization ineffectiveness was significantly higher in the third and in the fourth episode of gastroenteritis. The low efficacy of loperamide in our study could be related to a metabolic reaction due to drug interactions. Loperamide is mainly metabolized to desmethyl loperamide (DLOP) through the N-demethylation pathway [START_REF] Miyazaki | Disposition and metabolism of [14C]loperamide in rats[END_REF][START_REF] Yoshida | Metabolites of loperamide in rats[END_REF], and clinical studies have shown that DLOP formed by LOP N-demethylation is a major metabolite of LOP in humans [START_REF] He | Quantitation of loperamide and N-demethyl-loperamide in human plasma using electrospray ionization with selected reaction ion monitoring liquid chromatography-mass spectrometry[END_REF][START_REF] Tayrouz | Ritonavir increases loperamide plasma concentrations without evidence for Pglycoprotein. involvement[END_REF]. Moreover, it has been demonstrated that CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyze LOP N-demethylation in human liver microsomes, and among them, CYP2C8 and CYP3A4 may play a crucial role in loperamide N-demethylation at therapeutic concentrations [START_REF] Kim | Identification of cytochrome P450 isoforms involved in the metabolism of loperamide in human liver microsomes[END_REF]. Moreover, loperamide is a glycoprotein P substrate; therefore, its absorption in the bowel may be related to drug coadministration. Therefore, it is possible that drug interaction may modify loperamide elimination in humans. In fact, concurrent administration with CYP3A4 inhibitors such as fluvoxamine or erythromycin may lead to clinically significant reductions in plasma loperamide concentrations. Moreover, it is possible that the coadministration of a CYP3A4 enzyme inducer (such as carbamazepine or phenobarbital) may reduce loperamide effectiveness. In our study, all patients were in polytherapy with omeprazole, esomeprazole, antidepressants, or anitarrhythmics, which are inhibitors of CYP3A4 and glycoprotein P, as well as with phenobarbital, which is a CYP3A4 inductor. Therefore, we postulate that the loperamide ineffectiveness could be related to drug interaction. In contrast, racecadotril is a prodrug that is quickly metabolized by hydrolysis to its active metabolite, thiorphan, which in turn mediates enkephalinase inhibition [START_REF] Lecomte | Pharmacological properties of acetorphan, a parenterally active enkephalinases inhibitor[END_REF]. Therefore, in our study, racecadotril induced rapid remission of diarrhea (1-day treatment), because its action was not modified by polytherapy.

Adverse drug reactions (ADRs) represent a major public health problem in older populations [START_REF] Pirmohamed | Adverse drug reactions as cause of admission to hospital: prospective analysis of 18 820 patients[END_REF]. Bejer and de Blaey [START_REF] Beijer | Hospitalizations caused by adverse drug reactions (ADR): a meta-analysis of observational studies[END_REF] reported that the proportion of admissions related to ADRs in older people was four times higher than in younger people. In addition, we documented that ADRs are more common in elderly patients than in the young [START_REF] Gallelli | Retrospective evaluation of adverse drug reactions induced by nonsteroidal anti-inflammatory drugs[END_REF][START_REF] Gallelli | Adverse drug reactions to antibiotics observed in two pulmonology divisions of Catanzaro, Italy: a six-year retrospective study[END_REF][START_REF] Gallelli | Retrospective analysis of adverse drug reactions to bronchodilators observed in two pulmonary divisions of Catanzaro, Italy[END_REF][START_REF] Gallelli | Retrospective analysis of adverse drug reactions induced by gemcitabine treatment in patients with nonsmall cell lung cancer[END_REF]. It was recently reported that comorbidity could predict repeat admission for ADRs in older adults [START_REF] Zhang | Comorbidity and repeat admission to hospital for adverse drug reactions in older adults: retrospective cohort study[END_REF]. The mu-opiate-receptor agonist, loperamide, and other opiates, may cause adverse effects such as reactive constipation and abdominal distension [START_REF] Schiller | Mechanism of the antidiarrheal effect of loperamide[END_REF]. Their mode of action is through disrupting forward propulsive motility, increasing gut capacity, and delaying passage of fluid through the intestine [START_REF] Kachel | Human intestinal motor activity and transport: effects of a synthetic opiate[END_REF][START_REF] Sninsky | Effect of lidamidine hydrochloride and loperamide on gastric emptying and transit of the small intestine: a double-blind study[END_REF].

In agreement with another study [START_REF] Rouge | The enkephalinase inhibitor, acetorphan, in acute diarrhea: a double-blind control clinical trial versus loperamide[END_REF], we found that in racecadotril treatment, abdominal distension vanished rapidly, and reactive constipation was less frequent with respect to the loperamide group. These differences can be accounted for by the distinct mechanisms of antidiarrheal activity of the two drugs as above described. In both treatment groups, we documented the development of nausea, which was more frequently in the loperamide group than in the racecadotril group, but it did not cause treatment cessation. Efficacy and side effects may be related both to drug interaction and to genetic polymorphism. Recently, we documented that CYP2D6 polymorphism reduces the effectiveness of opiate agonists in polydrug abuse [START_REF] De Fazio | Role of CYP3A5 in abnormal clearance of methadone[END_REF]. To exclude the possibility that loperamide ineffectiveness could be related to gene polymorphism, we performed a pharmacogenetic analysis of CYP3A4 and CYP2C8 expression in unresponsive patients and in patients who developed side effects. Genetic analysis did not identify rapid or poor metabolizers; therefore, we can exclude that genetic susceptibility may be related to loperamide ineffectiveness or loperamide-related side effects in our analysis.

Side effects and drug ineffectiveness are responsible for a significant increase in direct costs during loperamide treatment. In fact, the direct cost of loperamide treatment was 367.95 euros compared with 179.4 euros for racecadotril treatment in ITT analysis. Moreover, the number of stool episodes was higher in the loperamide group compared with the racecadotril group, which influenced the decision switch all patients to racecadotril. Therefore, in PP analysis, loperamide treatment was not cost effective compared with racecadotril (430.05 euros for the loperamide group and 179.04 euros for the racecadotril group). This study was performed in nursing-home residents, so we assumed that indirect costs were similar in both groups.

In conclusion, we found that racecadotril is more effective than loperamide in elderly patients-which may be related to drug interaction with loperamide-and racecadotril is not dependent on pharmacogenetic susceptibility. It is also more cost effective than loperamide. Therefore, in elderly patients with diarrhea, racecadotril could be the first therapeutic option after ORS therapy to reduce the risk of dehydration.

Eur J Clin Pharmacol (2010) 66:137-144