Isabella Castellano

Elena Allia

Valeria Accortanzo

Anna Maria Vandone

Luigi Chiusa

Riccardo Arisio

Antonio Durando

Michela Donadio

Gianni Bussolati

Alan S Coates

Giuseppe Viale

MD Anna Sapino
email: anna.sapino@unito.it

Androgen receptor expression is a significant prognostic factor in estrogen receptor positive breast cancers

Keywords: Androgen receptor, prognosis, estrogen receptor, breast cancer, chemotherapy

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

Increasing data support a possible role of the androgen receptor (AR) as a marker of prognosis [START_REF] Rakha | Prognostic markers in triple-negative breast cancer[END_REF][START_REF] Schippinger | Evaluation of the prognostic significance of androgen receptor expression in metastatic breast cancer[END_REF], particularly among patients with estrogen receptor (ER) positive breast cancer [START_REF] Peters | Androgen receptor inhibits estrogen receptor-alfa activity and is prognostic in breast cancer[END_REF]. AR are frequently expressed together with ER and progesterone receptor (PgR) in well diffe rentiated breast cancer [START_REF] Moinfar | Androgen receptors frequently are expressed in breast carcinomas: Potential relevance to new therapeutic strategies[END_REF][START_REF] Kuenen-Boumeester | The clinical significance of androgen receptors in breast cancer and their relation to histological and cell biological parameters[END_REF][START_REF] Sapino | Expression of apocrine differentiation markers in neuroendocrine breast carcinomas of aged women[END_REF], but are rarely present in human epidermal growth factor receptor 2 (HER2) positive tumors [START_REF] Kollara | Loss of androgen receptor associated protein 70 (ARA70) expression in a subset of HER2-positive breast cancers[END_REF][START_REF] Ogawa | Androgen receptor expression in breast cancer: relationship with clinicopathological factors and biomarkers[END_REF]. Specific cancer histotypes, such as the apocrine carcinoma consistently express AR [START_REF] Gatalica | Immunohistochemical analysis of apocrine breast lesions. Consistent overexpression of androgen receptor accompanied by the loss of estrogen and progesterone receptors in apocrine metaplasia and apocrine carcinoma in situ[END_REF][START_REF] Niemeier | Androgen receptor in breast cancer: expression in estrogen receptor-positive tumors and in estrogen receptor-negative tumors with apocrine differentiation[END_REF] despite being ER and PgR-negative, and AR expression has been reported in about 45% of triple negative breast cancers [START_REF] Moinfar | Androgen receptors frequently are expressed in breast carcinomas: Potential relevance to new therapeutic strategies[END_REF][START_REF] Ogawa | Androgen receptor expression in breast cancer: relationship with clinicopathological factors and biomarkers[END_REF]. Studies using different analytical procedures suc h as a binding assay [START_REF] Bryan | Androgen receptors in breast cancer[END_REF], immunohistochemistry (IHC) [START_REF] Peters | Androgen receptor inhibits estrogen receptor-alfa activity and is prognostic in breast cancer[END_REF][START_REF] Agrawal | Androgen receptors as a prognostic and predictive factor in breast cancer[END_REF] and reverse-phase protein arrays [START_REF] Gonzalez-Angulo | Androgen receptor levels and association with PIK3CA mutations and prognosis in breast cancer[END_REF] have suggested a possible role of AR to predict responsiveness to endocrine therapy. The impact of AR expression on survival of patients with high risk ER-positive breast cancer, such as luminal B breast cancer [START_REF] Sorlie | Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications[END_REF][START_REF] Perou | Molecular portraits of human breast tumours[END_REF][START_REF] Sorlie | Repeated observation of breast tumor subtypes in independent gene expression data sets[END_REF], has never been evaluated. However, it has been shown that in metastatic breast cancers, the median survival after disease recurrence of patients with AR-expressing tumors was significantly longer compared to that of patients with AR-negative tumors [START_REF] Schippinger | Evaluation of the prognostic significance of androgen receptor expression in metastatic breast cancer[END_REF].

In this study, we evaluated the prognostic value of AR in a large series of patients with ER positive breast cancer after long follow-up. All patients were treated with endocrine therapy, with or without addition of chemotherapy. In particular, we focused on a subset of patients with tumors closely similar to luminal type B by conventional markers [START_REF] Cheang | Ki67 index, HER2 status, and prognosis of patients with luminal B breast cancer[END_REF] and considered as more clinically aggressive than the luminal type A ER-positive breast cancers.

Patients and methods

Study design

A consecutive series of 953 patients diagnosed with ER-positive breast cancer between 1998 and 2003 was retrieved from the files of the Pathology Departments of the San Giovanni Battista-Molinette Hospital (431 cases) and S. Anna Hospital (522 cases) in Turin, Italy. Tumor slides were centrally reviewed by two of the authors (AS, IC). Representative blocks were obtained and multicore tissue microarrays (TMAs) were prepared, as previously described [START_REF] Sapino | Routine assessment of prognostic factors in breast cancer using a multicore tissue microarray procedure[END_REF], using the advanced tissue arrayer (mod ATA-100, Chemicon International, Tamecula, CA, USA). Sections of the resulting recipient blocks were retested by IHC for ER status. Fifteen of 953 tumors were deemed ER-negative on TMAs and the negative results were further confir med on whole tissue sections. These cases were excluded from the study (Fig. 1). Clinical data of the remaining 938 patients including age, type of surgery, type of treatment, occurrence and type of relapse and current status were obtained from the Oncology Department of the two institutions. To confirm accuracy of previously recorded data, medical charts of all patients were reviewed. Pathological data including histotype, grade, and size of the tumors, peritumoral vascular invasion and stage were recorded according to the original diagnosis. The study was conducted in compliance with the ethical regulatory issues of the participating institutions.

Immunohistochemical procedure IHC was performed using an automated slide processing platform (Ventana BenchM ark AutoStainer, Ventana Medical Systems, Tucson, AZ, USA) and the following primary antibodies: prediluted anti-ER rabbit monoclonal antibody (SP1, Ventana-Diapath, Tucson, AZ, USA); prediluted anti-PgR rabbit monoclonal antibody (1E2, Ventana-Diapath); anti-AR mouse monoclonal antibody (AR441, diluted 1:50, Dako, Glostrup, Denmark); anti-Ki67 monoclonal antibody (MIB1, diluted 1:100 Dako) and anti-HER2 polyclonal antibody (A0485, diluted 1:800, Dako). FISH assays for assessing HER2 gene amplification were performed for IHC equivocal (score 2+) cases, as previously reported [START_REF] Marchiò | Does chromosome 17 centromere copy number predict polysomy in breast cancer? A fluorescence in situ hybridization and microarray-based CGH analysis[END_REF].

Positive and negative controls (omission of the primary antibody and IgG-matched serum) were included for each immunohistochemical run.

All TMAs slides were scanned by the Aperio system (ScanScope CS System, Vista, Ca, USA) and automated counting was performed. To ensure the reliability of the automatic assessment all cases were reviewed on screen by two of the authors ( A.S. and I.C.) blinded to patients' treatment assignment or outcome. If the recorded percentage of immunostained cells differed by more than 10%, then a collegial re-evaluation of the results was performed by three pathologists at the multiheaded microscope (I.C., R.A. and A.S.). This occurred for 1.2% of tumors. According to previous studies the cut off value for ER and PgR positivity was set at >1% and the same cut-off was also adopted for AR positivity [START_REF] Liu | Progesterone receptor is a significant factor associated with clinical outcomes and effect of adjuvant tamoxifen therapy in breast cancer patients[END_REF]. The percentage of Ki67-positive cells was recorded and the cut-off for dichotomizing tumors with low and high proliferative fraction was established at 10% positive cells for the overall study population, and at 14% for sorting out luminal type B breast cancer as suggested by Cheang MC et al [START_REF] Cheang | Ki67 index, HER2 status, and prognosis of patients with luminal B breast cancer[END_REF]. HER2 status was classified as negative or positive (when scored 3+ by IHC or HER2 amplified by FISH) according to the recommended guidelines for invasive carcinoma [START_REF] Wolff | American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer[END_REF].

Statistical Analysis

Differences between AR positive versus AR negative tumors were analyzed by univariate analysis with the Chi-square or Fisher's exact test: P-values<0.05 were considered to be statistically significant.

Median follow-up time was calculated as the median observation time among all patients. Follow-up was censored at the time of death or the last clinical investigation of the patient.

Disease-specific survival (DSS) was calculated from the data of definitive surgery to the date of death of disease. Patients dying from other causes were censored at the time of death. Time to relapse (TTR) was measured from the data of definitive surgery to the data of first recurrence (defined as loco-regional or systemic). Contralateral breast cancer was not considered as relapse.

The Kaplan-Meier method was used to estimate the TTR and DSS. The logrank test was used to test the survival differences.

Univariate analysis was used to examine what variables were associated with prognosis.

Variables of interest included: age, histotype, histological grade, pT and pN staging, vascular invasion, type of surgery (mastectomy versus breast conserving surgery), radiotherapy and chemotherapy. In order to adjust the risk estimate for potential confounders, a Cox proportional hazard regression for DSS and TTR was used and hazard ratios (HR) were calculated. Multivariate analysis included variables significant at P<0.05 in univariate models or with a priori hypothesis for inclusion.

Statistical analysis was carried out using the SPSS version 17 (SPSS Inc, Chicago, IL) software, the R environment [www.r-project.org], SAS version 9.1 (SAS Institute, Cary, NC) and S-PLUS version 6.1 (Insightful Corp, Seattle, WA). This article was written in accordance with Reporting Recommendations for Tumor Marker Prognostic Studies guidelines [START_REF] Mcshane | REporting recommendations for tumor MARKer prognostic studies (REMARK)[END_REF].

Results

Patients' characteristics

The study cohort included 938 patients with tumors confirmed as ER positive. Clinical and histopathological features of the whole population are reported in Table 1. The median follow-up time was 6.8 years.

All patients were treated with endocrine therapy. The majority received tamoxifen as first option, 180 patients switched from tamoxifen to an aromatase inhibitor after 2 years. Using the local protocol (Online Resource 1) defined during the time period of this study (patient's age <50 years and tumor >2 cm in size and/or presence of lymph node metastases and/or presence of peritumoral vascular invasion), a cohort of 390 patients received both endocrine and chemotherapy.

Androgen receptor expression and outcome AR was assessable in 859 tumors and 609 (70.9%) of these were considered positive. As shown in Table 2, AR positivity was associated with small tumor size (<2 cm), absence of lymph node metastases and PgR expression. Of the 250 AR-negative cases, 137 (54.8%) were treated with endocrine therapy only and 113 (45.2%) with endocrine and chemotherapy; of the 609 AR positive cases 358 (58.7%) were treated with endocrine therapy only and 251 (41.2%) with endocrine and chemotherapy.

Univariate analysis performed on the whole cohort of patients confirmed the correlation of the traditional histological and immunophenotypical factors with TTR (Table 3) and DSS (Table 4).

Focusing on AR expression, 609 patients with AR-positive tumors had a better outcome (Fig. 2a and 2b) (TTR: P=0.001 and DSS: P<0.001). Then we evaluated the effect of AR expression in the subcohort of patients treated with endocrine therapy alone. In this subset the P-value of AR positivity was marginally significant at 0.046 for TTR (Table 3) and not significant for DSS (P=0.247; Table 4; Fig. 3a). By contrast, in the group of patients receiving endocrine and chemotherapy, AR positivity was a strong prognostic factor for both TTR (P=0.015; Table 3) and DSS (P<0.001; Table 4, Fig. 3b). Following this result, we evaluated the effect of AR according to the different chemotherapeutic regimens, and we observed that AR expression was a marker of good prognosis in the group of patients treated with cyclophosphamide, methotrexate, and fluorouracil (CMF) (P=0.001) and anthracycline (P= 0.018) (Fig. 3c and3d). While no such significance was demonstrated among subsets of patients receiving other types of chemotherapy the numbers were limited and the data do not establish a reliable difference.

Cox multivariate analysis applied on the whole series of ER-positive cases for both TTR and DSS confirmed that AR status was an independent prognostic factor (Table 5) . The same role of AR was observed at Cox analysis for TTR, but not for DSS in patients treated with endocrine therapy alone (Table 6), while AR remained an independent factor of good prognosis for both TTR and DSS in the group of patients treated with endocrine and chemotherapy, as did PgR and HER2 (Table 7). However, a formal test for interaction between the use of chemotherapy and the expression of AR was not significant either for TTR (P=0.66) or DSS (P=0.21) (Fig. 4), so this apparent difference needs to be interpreted with caution.

Androgen receptor expression and outcome in luminal type B breast cancer

To further explore the prognostic value of AR in ER-positive breast cancer with more aggressive biology that may be the target of chemotherapy, we selected a series of 343 cases classified on the basis of immunohistochemical markers as closely similar to the genetically defined Luminal B, as recently proposed by Cheang M.C. et al [START_REF] Cheang | Ki67 index, HER2 status, and prognosis of patients with luminal B breast cancer[END_REF]. Briefly, we considered as Luminal B HER2 positive tumors and HER2 negative tumors with Ki67 >14%. Comparison of DSS and TTR of luminal type B against luminal A tumors confirmed poorer outcome among patients classified as Luminal B (Fig. 3e). Univariate analysis showed that the prognostic value on DSS of AR was maintained within the luminal B tumors (Fig. 3f) and Cox analysis confirmed this result for AR together with age, tumor size, vascular invasion and chemotherapy (Table 8).

Discussion

In the present study on a large series of ER-positive breast cancers, AR expression, as evaluated by IHC, provides statistically significant prognostic value beyond established clinicopathological parameters.

The role of AR as a prognostic marker has been suggested by other authors, though there is a high variability in patient populations, assay methods, analysis of results, definitions used and reporting of results [START_REF] Peters | Androgen receptor inhibits estrogen receptor-alfa activity and is prognostic in breast cancer[END_REF][START_REF] Niemeier | Androgen receptor in breast cancer: expression in estrogen receptor-positive tumors and in estrogen receptor-negative tumors with apocrine differentiation[END_REF][START_REF] Bryan | Androgen receptors in breast cancer[END_REF][START_REF] Agrawal | Androgen receptors as a prognostic and predictive factor in breast cancer[END_REF][START_REF] Park | Expression of androgen receptors in primary breast cancer[END_REF]. Particularly, the IHC cut-off values for positive and negative AR status are quite variable within different studies [START_REF] Peters | Androgen receptor inhibits estrogen receptor-alfa activity and is prognostic in breast cancer[END_REF][START_REF] Ogawa | Androgen receptor expression in breast cancer: relationship with clinicopathological factors and biomarkers[END_REF] or are not reported at all [START_REF] Agrawal | Androgen receptors as a prognostic and predictive factor in breast cancer[END_REF]. In the present study we used an automated system of analysis of IHC reactions that should avoid the bias of subjectivity linked to quantitative evaluation of IHC markers as performed by traditional microscopy. Using a cut off of >1%, the AR expression rate in ER-positive breast cancer was of almost 71%, which is comparable with previous reports on breast cancer in general [START_REF] Moinfar | Androgen receptors frequently are expressed in breast carcinomas: Potential relevance to new therapeutic strategies[END_REF][START_REF] Ogawa | Androgen receptor expression in breast cancer: relationship with clinicopathological factors and biomarkers[END_REF]. If the cutoff was increased to >10%, the prevalence of positive cases would decrease to 60%, though the significant correlation with outcome is maintained (Online Resource 2). A possible bias of the present work might be the use of TMAs, but using the multicore procedure we have previously shown that hormone receptors -and in particular ER levels-are correctly evaluated without significant discrepancy as compared to whole section analysis [START_REF] Sapino | Routine assessment of prognostic factors in breast cancer using a multicore tissue microarray procedure[END_REF].

In addition, we showed that positive AR expression by IHC identifies patients with better prognosis within the subset of luminal type B tumors. Gene expression studies have identified two molecularly distinct subtypes of ER-positive breast cancers, the luminal types A and B, with luminal B tumors having poorer outcomes than luminal A tumors [START_REF] Sorlie | Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications[END_REF][START_REF] Perou | Molecular portraits of human breast tumours[END_REF][START_REF] Sorlie | Repeated observation of breast tumor subtypes in independent gene expression data sets[END_REF]. Recent studies have suggested that a combination of conventional IHC markers [START_REF] Cheang | Ki67 index, HER2 status, and prognosis of patients with luminal B breast cancer[END_REF] may be considered a suitable surrogate for identifying a subset of breast cancers closely similar to the luminal B type defined by gene expression analysis. Our results show that in luminal type B cancers AR expression could be used as a prognostic marker, selecting a group of AR-positive tumors with better prognosis.

The likelihood of response of ER-positive breast cancers to adjuvant systemic chemotherapy cannot be accurately predicted, and the results of our retrospective study do not allow any conclusion that AR expression can predict which patients will benefit from the addition of chemotherapy, or indeed any particular type of chemotherapy, though the information that overall risk of relapse is lower in patients whose tumors express AR may assist decision-making in some cases. However, a recent study of triple negative breast cancers has shown by in vitro ATP-based chemotherapy response assay (ATP-CRA) that chemosensitivity to 5-fluorouracil and methotrexate was higher for tumors expressing AR and that this effect was not observed with other chemotherapeutics, such docetaxel, doxorubicin, epirubicin, vinorelbine, gemcitabine, oxaliplatin and paclitaxel [START_REF] Koo | The Predictive Role of E-cadherin and Androgen Receptor on In Vitro Chemosensitivity in Triple-negative Breast Cancer[END_REF]. Although a statistically significant interaction between chemotherapy and the expression of AR was not demonstrated, our results confirmed both by uni-and multivariate analysis a significant correlation of AR positive immunoreaction and longer DSS in patients treated with any chemotherapeutic regimen containing CMF and also with regimens containing an anthracycline, while there was not a significant correlation of AR with survival in patients treated with taxanes or vinorelbine. On the other hand, recent experimental data suggest a role for AR overexpression as a novel mechanism of tamoxifen resistance and speculate that AR and ER could collaborate to regulate cyclin D1 gene expression, thereby promoting cell cycle progression in the presence of tamoxifen [START_REF] De Amicis | Androgen receptor overexpression induces tamoxifen resistance in human breast cancer cells[END_REF]. Taken together these data may suggest that AR/ER positive breast cancer with high Ki67 expression could resist to tamoxifen therapy and benefit from specific chemotherapeutic treatment containing CMF and/or anthracyclines. Specific clinical trials and independent data set of patients with ER positive disease who received chemo-endocrine therapy are needed to confirm this hypothesis.

The 2009 St. Gallen recommendations consider endocrine therapy indicated if any ER

staining is present in the tumor [START_REF] Goldhirsch | Thresholds for therapies: highlights of the St Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer[END_REF]. One of the most difficult challenges in treating these patients, however, is the identification of patients with incompletely endocrine-responsive tumor who should receive a chemo-endocrine treatment. In prostate cancer, it has been suggested that knowledge of AR action in promoting cell proliferation can be used to design strategies that maximize cell death in response to cytotoxic therapy [START_REF] Hess-Wilson | Mitogenic action of the androgen receptor sensitizes prostate cancer cells to taxane-based cytotoxic insult[END_REF]. The results of the present study suggest that AR could be an important parameter for designing specific systemic treatments for the patients with ER-positive breast cancer.

Fig. 1

 1 Fig. 1 Study flowchart shows the process for patient selection

Fig. 2 Fig. 3 Fig. 4

 234 Fig. 2 Kaplan-Meier estimates of (a) time to relapse and (b) disease specific survival according to

Table 1

 1 Clinical and histopathological characteristics of 938 estrogen receptor positive breast cancers

		Nu mber	Total %	Valide %
		of cases
	Age
	< 40 years	31	3.3	3.3
	40-70 years	717	76.4	76.4
	> 70 years	190	20.3	20.3
	Relapse Type
	No	791	84.3	84.3
	Local	35	3.7	3.7
	Systemic	90	9.6	9.6
	Controlateral	22	2.3	2.3
	Death of disease
	No	868	92.5	92.5
	Yes	70	7.5	7,5
	Histotype
	Ductal and Lobular	638	68.0	68.0
	Special types	300	31.9	31.9
	Grade
	1	315	33.6	33.6
	2	406	43.3	43.3
	3	217	23.1	23.1
	pT
	1	600	64,0	64,0
	2	282	30.1	30.1
	3	26	2.8	2.8
	4	29	3.1	3.1
	x	1	0.1
	pN
	0, ITC, 1MIC	508	54.2	62.0
	1,2,3	311	33.2	38.0
	Unknown	119	12.7
	Type of surgery
	Mastectomy	255	27.2	27.2
	Conservative	683	72.8	72.8
	Vascular invasion
	No	429	45.7	45.7
	Yes	509	54.2	54.2
	Radiotherapy
	No	258	27.5	27.5
	Yes	680	72.5	72.5
	Chemotherapy
	No	548	58.4	58.4
	Yes	390	41.6	41.6
	Type of endocrine therapy
	Tamo xifen	709	75.5	75.5
	Switch	180	19.1	19.1
	Aromatase Inhibitors	49	5.2	5.2
	Type of chemotherapy
	CMF	112	28.7	28.7
	Anthracycline	66	16.9	16.9
	FEC or CMF+FEC	116	29.7	29.7
	Taxanes or Vinorelbine	96	24.6	24.6

ITC isolated tumor cells; MIC micrometastasis; CMF cyclophosphamide, methotrexate and fluorouracil; FEC fluorouracil, epirubicin and cyclophosphamide

Table 2

 2 Patients' and histopathological characteristics according to androgen receptor status ITC isolated tumor cells; MIC micrometastasis; PgR Progesterone Receptor, HER 2 human epidermal growth factor receptor 2.

	Characteristics	AR	AR	2	P-value
		negative	positive
	Age
	< 40 years	6	23
	40-70 years	194	460	1.336	0.567
	> 70 years	50	126
	Histotype
	Ductal and Lobular	163	419	0.894	0.335
	Special types	87	190
	Grade
	Low	82	201
	Intermediate	102	277	2.731	0.255
	High	66	131
	pT
	1	147	394
	2	79	186	8.42	0.038
	3	9	16
	4	14	13
	pN
	0, ITC, 1MIC	115	347	8.656	0.003
	1,2,3	101	186
	Vascular invasion
	No	109	275	0.116	0.733
	Yes	141	334
	PgR status
	<1%	65	114	5.263	0.022
	>1%	185	495
	Ki67
	<10%	91	257	1.212	0.271
	>10%	117	272
	HER 2
	Negative	221	525	1.791	0.408
	Positive	26	74
	AR Androgen Receptor;

Table 3 .

 3 Univariate analysis of clinical and pathological data correlated with time to relapse.

	CPS Cumulative Proportion Surviving; n.d. not done; ITC isolated tumor cells; MIC
	micrometastasis, PgR Progesterone Receptor; HER2 human epidermal growth factor receptor 2;
	AR Androgen Receptor.

Table 4

 4 Univariate analysis of clinical and pathological data correlated with disease specific survival

	General		Endocrine therapy	Endocrine and chemotherapy
	General CPS CPS at 10 years (range 0-1) Censored % Censored % at 10 years (range 0-1) 80.6 0.82 67.7 0.74 93.7 0.89 86.6 0.79 90 0.77 88.4 0.81 91.9 0.86 96.1 0.92 84.9 0.78 74.6 0.66 90.8 0.83 96 0.91 83.1 0.77 74.5 0.68 84.3 0.75 91.1 0.85 95.7 0.93 90.7 0.87 96.8 0.93 93.6 0.91 93.8 0.89 88.9 0.80 83.9 0.76 71 0.63 96.3 0.92 91.2 0.84 80.8 0.72 87.9 0.77 73.1 0.64 73.1 0.71 75.9 0.78 51.7 0.42 Vascular invasion Age (years) < 40 Age (years) < 40 40-70 40-70 > 70 > 70 pN Pn 0, ITC, 1MIC 0, itc, 1mic 1,2,3 1,2,3 Type of surgery Type of surgery Mastectomy Mastectomy Conservative Conservative Histotype Histotype Ductal and Lobular Ductal and Lobular Special types Special types Grade 1 Grade 1 2 2 3 3 pT 1 pT 1 2 2 3 3 4 4 Vascular invasion No 93.5 0.83 No 97.4 0.94 Yes 88.4 0.82 Yes 80.3 0.74 Radiotherapy Radiotherapy No 79.8 0.72 No 87.2 0.90 Yes 94.5 0.80 Yes 88.8 0.81 PR PgR 0 85,6 0,78 0 93.4 0.81 >1% 89.5 0.88 >1% 86,7 0,78 HER2 HER2 Neg 87.4 0.79 Neg 93 0.87 Pos 90.1 0.89 Pos 79.3 0.72 AR AR 0 80 0.69 0 86.4 0.78 >1% 95.2 0.92 >1% 89.3 0.83 KI67 KI67 0 93.8 0.86 >1% 74.1 0.60 0 97.3 0.95 >1% 86.7 0.85	P-value P-value 0.006 0.002 <0.001 <0.001 <0.001 <0.001 0.023 0.037 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.001 0.001 0.469 0.019 0.011 0.176 0.001 <0.001 <0.001 0.002	Endocrine therapy CPS Censored % CPS at 10 years (range 0-1) P-value % Censored at 10 years (range P-value 0-1) 80 n.d. 100 n.d 0.571 0.008 95.8 0.93 91.8 0.88 92.1 0.79 91.5 0.83 92.6 0.87 0.006 95.8 0.92 <0.001 81.7 0.72 81.7 0.77 94.4 0.88 <0.001 97.4 0.93 <0.001 84.4 0.80 82.8 0.78 89.6 0.84 0.039 92.3 0.87 0.011 98.1 0.97 95.2 0.92 97.4 0.98 95.2 0.93 0.004 0.002 93.7 0.88 90.4 0.83 85.1 0.79 82.1 0.76 97.2 0.94 94.1 0.89 <0.001 86.7 0.82 <0.001 87.6 0.77 66.7 n.d. 77.8 n.d. 80 n.d. 60 n.d. 95.6 0.89 <0.001 97.6 0.95 <0.001 89.5 0.83 85.7 0.82 85.8 0.81 0.004 88.1 0.93 <0.001 96.6 0.84 93.7 0.89 91.9 0.91 0.826 93 0.91 0.250 95.1 0.92 92 0.87 91.8 0.87 0.958 94.4 0.89 0.654 96 0.95 92 0.85 88.3 0.76 0.046 92.7 0.83 0.247 95.5 0.93 93.8 0.93 97.2 0.93 <0.001 80.3 0.74 98.4 0.96 0.002 87.3 0.80	CPS Endocrine and chemotherapy % Censored CPS at 10 years % Censored at 10 P-years value (range P-value 0-1) (range 0-1) 80.8 0.83 61.5 0.003 91.4 0.85 0.58 0.010 80.8 76.9 n.d. 0.69 69.2 n.d. 89.8 96.9 0.90 0.005 85.7 0.80 0.81 0.001 72.9 0.63 84.8 0.75 93.8 0.89 0.002 82 0.74 <0.001 66.9 0.57 78.3 89.7 0.82 0.954 0.66 90 0.85 0.796 80 0.78 92.6 0.78 83.3 0.005 93.9 0.91 0.79 <0.001 87.4 83.3 0.75 0.77 66 0.55 94.3 0.91 84.1 0.74 <0.001 77.4 88.1 0.75 0.007 76.5 0.72 0.66 70.6 73.7 0.78 0.70 47.4 0.42 85.6 96.7 0.92 0.018 87.7 0.80 0.65 0.066 76.7 0.69 73.4 86.3 0.76 0.184 91.3 0.87 0.63 0.097 81.2 0.71 78 85.4 0.71 0.037 91.2 0.86 0.66 0.611 79.1 0.68 81 90.8 0.83 0.064 85.2 0.84 0.70 0.013 68.8 0.62 69.9 78.8 0.72 <0.001 94.8 0.89 0.60 0.015 82.9 0.71 87 68.2 0.45 0.007 0.70 95.1 0.92 0.178 86.1 0.80

HR Hazard Ratio; CI Confidence Interval; PgR Progesterone Receptor, HER2 human epidermal growth factor receptor 2; AR Androgen Receptor.

Table 7

 7 Association of patient and tumor characteristics with time to relapse and disease specific survival among patients treated by endocrine and chemotherapy with co mplete data for all covariates

			PATIENTS TREATED BY ENDOCRINE THERA PY
			Time to relapse			Disease specific survival
		HR	95% CI	2	P-value	HR	95% CI	2	P-value
	Age	0.241 0.091-0.642 8.102 0.004	0.399 0.111-1.426 2.000	0.157
	pN	1.925 0.658-5.626 1.431 0.232	1.877 0.544-6.472 0.993	0.319
	Type of surgery	2.161 0.613-7.624 1.435 0.231	3.263 0.751-14.188 2.488	0.115
	Histotype	1.718 0.677-4.364 1.297 0.255	0.968 0.251-3.726 0.002	0.962
	Grade	0.918 0.518-1.629 0.085 0.771	1.038 0.498-2.166 0.010	0.921
	pT	1.838 1.079-3.131 5.023 0.025	2.410 1.285-4.522 7.508	0.006
	Vascular Invasion 3.738 1.367-10.221 6.599 0.010	3.564 0.946-13.423 3.527	0.060
	Radiotherapy	0.596 0.176-2.022 0.689 0.407	0.934 0.238-3.664 0.009	0.922
	PGR	1.042 0.355-3.063 0.006 0.940	0.506 0.150-1.703 1.210	0.271
	HER2	0.912 0.234-3.553 0.018 0.894	0.741 0.143-3.855 0.127	0.722
	AR	0.427 0.190-0.959 4.246 0.039	0.413 0.150-1.137 2.928	0.087
	KI67	2.684 0.969-7.438 3.604 0.058	1.566 0.477-5.142 0.546	0.460
			PATIENTS TREATED BY ENDOCRINE AND CHEM OTHERAPY
			Time to relapse			Disease specific survival
		HR	95% CI	2	P	HR	95% CI	2	P
	Age	0.489 0.253-0.947 4.505 0.034 0.280 0.091-0.866 4.881 0.027
	pN	1.721 0.849-3.486 2.269 0.132 1.367 0.362-5.170 0.213 0.645
	Type of surgery	1.990 0.966-4.096 3.486 0.062 1.666 0.590-4.706 0.927 0.336
	Histotype	1.376 0.752-2.516 1.074 0.300 2.353 0.853-6.494 2.730 0.098
	Grade	2.030 1.218-3.385 7.372 0.007 2.281 0.978-5.318 3.645 0.056
	pT	1.625 1.190-2.220 9.327 0.002 2.220 1.354-3.641 9.992 0.002
	Vascular Invasion	1.969 0.914-4.242 2.994 0.084 11.298 1.847-69.091 6.887 0.009
	Radiotherapy	1.062 0.529-2.131 0.029 0.865 0.652 0.234-1.822 0.664 0.415
	PgR	0.466 0.248-0.878 5.589 0.018 0.176 0.068-0.455 12.883 0.000
	HER2	2.211 1.216-4.022 6.758 0.009 3.022 1.138-8.021 4.928 0.026
	AR	0.444 0.258-0.765 8.555 0.003 0.135 0.054-0.337 18.339 0.000
	KI67	1.842 0.958-3.543 3.350 0.067 1.575 0.548-4.526 0.710 0.399

HR Hazard Ratio; CI Confidence Interval; PgR Progesterone Receptor, HER2 human epidermal gro wth factor receptor 2; AR Androgen Receptor.

Table 8

 8 Association of patient and tumor characteristics with time to relapse and disease specific survival among 343 patients with luminal B cancer with complete data for all covariates HR Hazard Ratio; CI Confidence Interval; PgR Progesterone Receptor; AR Androgen Receptor.

			PATIENTS WITH LUM INA L B BREA ST CANCER
			Time to relapse			Disease specifc survival
		HR	95%CI	2	P-value HR	95%CI	2	P-value
	Age	0.290 0.162-0.520 17.242 0.000 0.354 0.148-0.846	0.846	0.019
	pN	1.638 0.846-3.169 2.143 0.143 2.391 0.871-6.562	2.864	0.091
	Type of surgery	1.763 0.810-3.835 2.044 0.153 2.525	0.923-6.905	3.256	0.071
	Histotype	1.367 0.771-2.425 1.145 0.285 1.081 0.434-2.694	0.028	0.867
	Grade	1.197 0.791-1.810 0.724 0.395 1.130	0.625-2.043	0.163	0.686
	pT	1.826 1.367-2.439 16.595 0.000 2.008 1.339-3.011	11.377	0.001
	Vascular Invasion	2.203 1.093-4.438 4.884 0.027 6.836 1.737-26.908	7.560	0.006
	Radiotherapy	0.753 0.353-1.607 0.539 0.463 1.009 0.395-2.581	0.000	0.985
	Chemotherapy	0.780 0.398-1.530 0.521 0.470 0.351 0.142-0.870	5.114	0.024
	PgR	0.712 0.381-1.328 1.142 0.285 0.351 0.206-1.066	3.270	0.071
	AR	0.521 0.306-0.888 5.743 0.017 0.276 0.130-0.588	11.135	0.001

Acknowledgme nts of research support for the study: This study was funded by Compagnia San Paolo di Torino, Cassa di Risparmio di Torino, AIRC 2007-2009, Ricerca Finalizzata Regione Piemonte 2008.

CPS Cu mu lative Proportion Surviv ing; n.d. not done; ITC isolated tumor cells; MIC micro metastasis, PgR Progesterone Receptor; HER2 human epidermal growth factor receptor 2; AR Androgen Receptor.


N Fortunati

M G Catalano

F Marano

V Mugoni

M Pugliese

O Bosco

F Mainini

G Boccuzzi

Giovanni Battista

Prof Giuseppe Boccuzzi

The pan-DAC inhibitor

Keywords: DAC-inhibitors, breast cancer, NIS, targeted therapy, epigenetic

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

INTRODUCTION

Breast cancer is the most common cancer in women all over the world [START_REF] Parkin | Global cancer statistics[END_REF]. Even though its management has become more and more successful over time, owing to the different available therapeutic strategies [START_REF] Berry | Cancer Intervention and Surveillance Modeling Network (CISNET) Collaborators. Effect of screening and adjuvant therapy on mortality from breast cancer[END_REF], both metastatic disease and cancer which are becoming resistant to conventional treatment are emerging problems [START_REF] Gonzalez-Angulo | Overview of resistance to systemic therapy in patients with breast cancer[END_REF][START_REF] Cardoso | International guidelines for management of metastatic breast cancer: combination vs sequential single-agent chemotherapy[END_REF]. New drugs with anti-tumour activity and/or able to modify the expression of selected molecules in these situations are under evaluation.

The sodium/iodide symporter (NIS) has been receiving a good deal of attention, both for its function that is requisite for potential clinical application, and for its sensitivity to a number of agents able to induce/increase its cell expression. NIS is a transmembrane glycoprotein mainly expressed in thyrocytes, functioning as iodide carrier [START_REF] Dai | Cloning and characterization of the thyroid iodide transporter[END_REF]. The expression and correct functioning of NIS in differentiated thyroid cancer is the molecular requisite for successful radioiodide therapy in this neoplasm [START_REF] Reiners | Radio-iodine therapy in differentiated thyroid cancer: indications and procedures[END_REF]. NIS is also expressed in lactating mammary glands, favouring iodide concentration in milk, and in more than 80 % of human breast carcinomas [START_REF] Tazebay | The mammary gland iodide transporter is expressed during lactation and in breast cancer[END_REF]. Although NIS expression is present and intense in a majority of invasive breast cancers, radioiodide accumulation has been observed in only a small fraction of cases [START_REF] Wapnir | Immunohistochemical profile of the sodium/iodide symporter in thyroid, breast, and other carcinomas using high density tissue microarrays and conventional sections[END_REF][START_REF] Wapnir | The Na+/I-symporter mediates iodide uptake in breast cancer metastases and can be selectively down-regulated in the thyroid[END_REF][START_REF] Moon | Correlation between 99mTc pertechnetate uptakes and expressions of human sodium iodide symporter gene in breast tumor tissues[END_REF]. The achievement of a good level of expression of NIS is regarded as a promising new strategy to unable the use of radioiodide for imaging and treatment of breast cancer [START_REF] Boelaert | Sodium iodide symporter: a novel strategy to target breast, prostate, and other cancers?[END_REF][START_REF] Kogai | Differential regulation of sodium/iodide symporter gene expression by nuclear receptor ligands in MCF-7 breast cancer cells[END_REF].

In non neoplastic breast cells, estrogens, oxitocyn and prolactin, mainly acting in concert, were reported to induce NIS [START_REF] Tazebay | The mammary gland iodide transporter is expressed during lactation and in breast cancer[END_REF]. In breast cancer cells, many authors reported that retinoic acid is a powerful enhancer of NIS expression especially in association with corticosteroids [START_REF] Kogai | Differential regulation of sodium/iodide symporter gene expression by nuclear receptor ligands in MCF-7 breast cancer cells[END_REF][START_REF] Willhauck | Functional sodium iodide symporter expression in breast cancer xenografts in vivo after systemic treatment with retinoic acid and dexamethasone[END_REF][START_REF] Ohashi | Activation of the PI3 kinase pathway by retinoic acid mediates sodium/iodide symporter induction and iodide transport in MCF-7 breast cancer cells[END_REF]. Furthermore, in one report it was also observed that retinoic acid and dexamethasone treatment determined a significant 131 I-induced cytotoxicity in breast cancer cells [START_REF] Unterholzner | Dexamethasone stimulation of retinoic Acid-induced sodium iodide symporter expression and cytotoxicity of 131-I in breast cancer cells[END_REF]. NIS expression, in both thyroid and non-thyroid cells, is also regulated by epigenetic phenomena. NIS gene silencing can be related to the level of histone acetylation; in fact, compounds like Trycostatin A (TSA), sodium butyrate (NaB) and valproic acid (VPA), that belong to the decetylase inhibitor (DAC-inhibitor) group, stimulate NIS expression in thyroid cancer cells [START_REF] Kitazono | Low concentrations of the histone deacetylase inhibitor, depsipeptide (FR901228), increase expression of the Na(+)/I(+) symporter and iodine accumulation in poorly differentiated thyroid carcinoma cells[END_REF][START_REF] Zarnegar | Increasing the effectiveness of radioactive iodine therapy in the treatment of thyroid cancer using Trichostatin A, a histone deacetylase inhibitor[END_REF][START_REF] Fortunati | Valproic acid induces the expression of the Na+/I+ symporter and iodine uptake in poorly differentiated thyroid cancer cells[END_REF] DAC-inhibitors [START_REF] Botrugno | Histone deacetylase inhibitors as a new weapon in the arsenal of differentiation therapies of cancer[END_REF][START_REF] Lee | Histone deacetylase inhibitors in cancer therapy[END_REF] could also be used to control NIS expression inducing radioiodine sensitivity in different human extra-thyroid tumours such as breast cancer.

TSA and NaB greatly increased NIS mRNA also in breast cancer MCF-7 cell line [START_REF] Puppin | Effects of histone acetylation on sodium iodide symporter promoter and expression of thyroid-specific transcription factors[END_REF].

The aim of the present work was to evaluate the ability of the powerful pan-DAC inhibitor LBH589 (panobinostat) [START_REF] Atadja | Development of the pan-DAC inhibitor panobinostat (LBH589): successes and challenges[END_REF] to induce the expression of functional NIS in breast cancer cells with different estrogen sensitivity.

MATERIALS AND METHODS

Cell lines and reagents

Estrogen receptor-positive MCF-7 and T47D and estrogen receptor-negative MDA-MB 231 cells were purchased from ECACC (Salisbury, UK). corresponds to the concentration of the compound that inhibits 50% net cell growt h; TGI value is the concentration of the compound leading to total growth inhibition; LC 50 value is the concentration of the compound leading to 50% net cell death.

Apoptosis Detection -Cell Death Detection ELISA

For apoptosis studies, 4 x 10 3 cells were seeded in 96-well plates and treated with LBH589, using the same schedule as for the viability assay. After treatment, apoptosis was evaluated using Cell Death Detection ELISA PLUS (Roche Applied Science, Penzberg, Germany) following manufacturer's instructions. Apoptosis was expressed as enrichment factor, calculated as a fraction of the absorbance of treated cells versus untreated controls.

Cell cycle analysis

Cells were treated with LBH589 up to 72 hours. At different times (24, 48 and 72 hours), all cells were collected, fixed in 70% ethanol for 30 minutes on ice and incubated in propidium iodide solution (20 g/ml propidium iodide, 0.2 mg/ml RNAseA in PBS) for 1 hour at room temperature. The cell population was analyzed by flow cytometry.

Gene expression evaluation with Real Time PCR

Cells (1x10 6 ) were seeded in 75cm 2 flasks and treated with LBH589 (5-100nM).

Total RNA was extracted using TRIzol Reagent (Invitrogen Ltd, Paisley, UK), as previously described. DNase I was added to remove remaining genomic DNA. 1 g of total RNA was reverse-transcribed with iScript cDNA Synthesis Kit (BioRad Laboratories, Inc.), following manufacturer's protocol.

Primers (Table 1) were designed using Beacon Designer 5.0 software according to parameters outlined in the BioRad iCycler Manual. Specificity of primers was confirmed by BLAST analysis. Real-time PCR was performed using a BioRad iQ iCycler Detection System (BioRad Laboratories, Inc.) with SYBR green fluorophore. Reactions were performed in a total volume of 25 l, including 12.5 l IQ SYBR Green Supermix (BioRad Laboratories, Inc.), 1 l of each primer at 10 M concentration, and 5 l of the previously reversetranscribed cDNA template. Protocol for primer set was optimised using seven serial 5X dilutions of template cDNA obtained from cells in basal conditions. The protocol used is as follows: denaturation (95°C for 5 min), amplification repeated 40 times (95°C for 15 sec, 60°C for 1 min). A melting curve analysis was performed following every run to ensure a single amplified product for every reaction. All reactions were carried out at least three times for each sample.

Luciferase Transcription Assay

Cells (2.5 x 10 5 ) were seeded in 6-well plates. 24 hours after seeding, cells were transfected with 1μg of NIS-luc plasmid/well using Lipofectin Reagent (Invitrogen Ltd, Paisley, UK).

The plasmid, a kind gift of Prof. G. Damante (Dipartimento di Scienze & Tecnologie Biomediche, Policlinico Universitario di Udine, Udine) contains 2.2 kb of 5' genomic sequence for the NIS promoter and the minimal promoter is linked to the luciferase (LUC) gene as reporter [START_REF] Behr | Cloning of a functional promoter of the human sodium/iodide-symporter gene[END_REF]. After 24 hours, cells were treated with LBH589 (10-50 nM) for a further 24 hours. At completion, luciferase activity was assayed with the Luciferase Assay System (Promega Corporation, Madison, WI, USA).

NIS Immunoflorescence microscopy

Cells (5 x 10 5 ) were seeded in 6-well plates. 24 hours after seeding, cells were treated with 25 nM LBH589 for 48 hours. After treatment, cells were fixed in acetone/methanol (1:1) at -20 °C for 20 minutes, washed with PBS containing 0.5% Triton X-100, 0.05% NaN 3 and 1%

Horse Serum and incubated with monoclonal anti-NIS antibody (1:1000) (FP5A, Thermo

Fisher Scientific, Fremont CA, USA) in PBS at 4°C overnight. Then cells were washed with PBS containing 0.5% Triton and 0.05% NaN 3 for 10 minutes for three times followed by detection with anti-mouse Cy3-conjugated secondary antibody (1:1000), (GE Healthcare Europe, GmbH, Milano, Italy) in PBS plus 0.5% Triton and 0.05% NaN 3 for 2 hours and nuclei counterstaining with Hoechst 33258 (500 ng/ml in DMSO) in PBS. Cells were washed twice with distilled water and mounted with 50% glycerol-PBS media.

I uptake by cells

Cells (2 x 10 4 ) were seeded into 24-well plates and incubated with LBH589 (5-100nM) for 48 hours. 125 I uptake was assayed as reported elsewhere [START_REF] Fortunati | Valproic acid induces the expression of the Na+/I+ symporter and iodine uptake in poorly differentiated thyroid cancer cells[END_REF]. Briefly, after aspirating drugcontaining medium, cells were washed with 1 ml HBSS (Invitrogen, Groningen, The Netherlands). 125 I uptake was initiated by adding 0.5 ml HBSS containing 2 Ci carrier-free Na 125 I (PerkinElmer Life and Analytical Sciences, Inc., Boston, USA) and 30 M NaI.

Incubation was conducted for 30 min at 37°C and terminated by removing the radioactive medium and washing the cells twice with ice-cold HBSS. Cells were then solubilized with 1 ml of absolute ethanol for 20 min, after which cell-associated iodide was measured in acounter.

Statistical analysis

Data are expressed throughout as means SEM, calculated from at least three different experiments. Comparison between groups was performed with analysis of variance (one-way ANOVA) and the threshold of significance was calculated with the Bonferroni test. Statistical significance was set at P<0.05.

RESULTS

Anti-tumor activity of LBH589

LBH589 acts as deacetylase inhibitor (DAC inhibitor) in breast cancer cells, inducing hyperacetylation of histone H4, as reported in figure 1, and is a powerful cytotoxic agent causing a significant reduction in cell viability of MCF7, T47D, and MDA-MB231 cells (figure 2).

Furthermore, in figure 2 we report the calculated values of GI 50 , TGI, LC 50 , that are all in nanomolar range.

The mechanisms of action elicited by LBH589 to exert its cytotoxic activity in breast cancer cells are reported in figure 3. Actually, LBH589 was able to induce apoptosis in a dosedependent manner (panels on the left), and a significant growth arrest in G 2 /M (panels on the right). These effects were observed both in ER-positive and ER-negative breast cancer cells, even though higher doses of LBH589 were required in MDA-MB 231.

Effect of LBH589 on NIS Expression and Function

The ability of LBH589 to induce the Na + /I symporter in breast cancer cells was thoroughly evaluated.

NIS was first studied as mRNA expression. As reported in figure 4 (panels on the left), the expression of NIS mRNA was significantly induced by LBH589 in a dose-dependent manner in all the three breast cancer cell lines. In addition, in figure 4 (panels on the right) we report the effect of LBH589 on the activity of NIS promoter, evaluated with a luciferase assay, demonstrating that LBH589 is effective in inducing NIS promoter activity in the cell lines under study.

The induction of NIS gene transcription has to go together with the presence of NIS protein in cells, and this was clearly demonstrated in MCF7, T47D, and MDA-MB231 breast cancer cells under LBH589 treatment as shown in figure 5 (upper panels). Moreover, the symporter induced by LBH589 confers the ability of up-taking iodide to breast cancer cells (figure 5, lower panels).

In all these experiments, LBH589 was effective in inducing a functional NIS either in ERpositive and ER-negative breast cancer cells, even though ER-positive cells were shown to be more sensitive to the compound.

DISCUSSION

The present study shows the potent cytotoxic activity of LBH589 in both estrogen-sensitive and -insensitive cells and, especially, highlights its ability to induce the sodium/iodide symporter (NIS) in breast cancer cells that, as a consequence, become able to concentrate radioactive iodide.

At present four different trials are recruiting patients with recurrent or metastatic breast cancer for treatment with LBH589 both in monotherapy and in association with trastuzumab and paclitaxel (www.clinicaltrials.gov), although knowledge concerning the precise mechanism of LBH589 cytotoxicity in breast cancer cells is still incomplete. The primary molecular effect of DAC-inhibitors is to alter the acetylation status of the core histone proteins, facilitating chromatin remodelling and modification in gene expression [START_REF] Bolden | Anticancer activities of histone deacetylase inhibitors[END_REF]. It has been reported that 100 and 200 nM LBH589 alone or in association with 4OHTamoxifene were effective in reducing the growth of ER-negative MDA-MB 231cells [START_REF] Zhou | Histone deacetylase inhibitor LBH589 reactivates silenced estrogen receptor alpha (ER) gene expression without loss of DNA hypermethylation[END_REF] and that LBH589 significantly reduced the viability of ER-positive MCF-7 cells [START_REF] Fiskus | Hydroxamic acid analogue histone deacetylase inhibitors attenuate estrogen receptor-alpha levels and transcriptional activity: a result of hyperacetylation and inhibition of chaperone function of heat shock protein 90[END_REF] and increased the fraction of dying cells after radiation exposure [START_REF] Kim | Epigenetic modulation of radiation response in human cancer cells with activated EGFR or HER-2 signaling: potential role of histone deacetylase 6[END_REF]. Here we demonstrate that LBH589 alone, in nanomolar range which is far from toxic range in humans [START_REF] Giles | A phase I study of intravenous LBH589, a novel cinnamic hydroxamic acid analogue histone deacetylase inhibitor, in patients with refractory hematologic malignancies[END_REF], is able to reduce the viability of ER-positive MCF-7

and T47D and ER-negative MDA-MB 231 breast cancer cells. Besides this, we show that the mechanisms underlying its effect on cell viability include both apoptosis induction and cell cycle arrest, as already reported for other DAC-inhibitors [START_REF] Fortunati | Valproic acid is a selective antiproliferative agent in estrogensensitive breast cancer cells[END_REF].

We demonstrate in particular that LBH589 induces and enhances NIS expression in breast cancer cells. The efficiency of NIS is at the basis of successful treatment of differentiated thyroid cancer thus the possibility of increasing NIS expression in thyroid neoplasms which have lost the ability to uptake and concentrate iodide is under extensive evaluation [START_REF] Dohán | The sodium/iodide Symporter (NIS): characterization, regulation, and medical significance[END_REF][START_REF] Furuya | Histone deacetylase inhibitors restore radioiodide uptake and retention in poorly differentiated and anaplastic thyroid cancer cells by expression of the sodium/iodide symporter thyroperoxidase and thyroglobulin[END_REF].

Similarly, as NIS is expressed in quite a significant percentage of breast cancers [START_REF] Tazebay | The mammary gland iodide transporter is expressed during lactation and in breast cancer[END_REF], NISmediated radionuclide imaging and targeted therapy could be useful goals in breast cancer management. Unfortunately, although NIS expression is present and intense in the majority of invasive breast cancers, only a small fraction of NIS-positive breast cancers are able to concentrate iodide, due to low cell surface NIS levels and cell surface trafficking defects, commonly found in breast tumours [START_REF] Beyer | Do cell surface trafficking impairments account for variable cell surface sodium iodide symporter levels in breast cancer?[END_REF]. Therefore, research efforts should focus on strategies to induce/increase NIS expression in breast cancer as it has to be adequately expressed in order to get a considerable iodide uptake. A large number of agents with very different mechanisms of action have been utilized to stimulate NIS expression, such as redifferentiating agents [START_REF] Kogai | Differential regulation of sodium/iodide symporter gene expression by nuclear receptor ligands in MCF-7 breast cancer cells[END_REF][START_REF] Unterholzner | Dexamethasone stimulation of retinoic Acid-induced sodium iodide symporter expression and cytotoxicity of 131-I in breast cancer cells[END_REF][START_REF] Kogai | Retinoic acid stimulation of the sodium/iodide symporter in MCF-7 breast cancer cells is mediated by the insulin growth factor-I/phosphatidylinositol 3-kinase and p38 mitogen activated protein kinase signaling pathways[END_REF][START_REF] Dohán | Hydrocortisone and purinergic signaling stimulate sodium/iodide symporter (NIS)-mediated iodide transport in breast cancer cells[END_REF][START_REF] Tanosaki | Effect of ligands of nuclear hormone receptors on sodium/iodide symporter expression and activity in breast cancer cells[END_REF], or compounds that are reported to physiologically regulate NIS in breast cells [START_REF] Tazebay | The mammary gland iodide transporter is expressed during lactation and in breast cancer[END_REF][START_REF] Cho | Hormonal regulation of radioiodide uptake activity and NaC/IK symporter expression in mammary glands[END_REF][START_REF] Arturi | Regulation of iodide uptake and sodium/iodide symporter expression in the MCF-7 human breast cancer cell line[END_REF][START_REF] Knostman | PI3K activation is associated with intracellular sodium/iodide symporter protein expression in breast cancer[END_REF]. Retinoid acid, especially in the presence of other compounds such as dexamethasone, induce NIS and iodide uptake in breast cancer cells [START_REF] Kogai | Differential regulation of sodium/iodide symporter gene expression by nuclear receptor ligands in MCF-7 breast cancer cells[END_REF][START_REF] Unterholzner | Dexamethasone stimulation of retinoic Acid-induced sodium iodide symporter expression and cytotoxicity of 131-I in breast cancer cells[END_REF][START_REF] Kogai | Retinoic acid stimulation of the sodium/iodide symporter in MCF-7 breast cancer cells is mediated by the insulin growth factor-I/phosphatidylinositol 3-kinase and p38 mitogen activated protein kinase signaling pathways[END_REF][START_REF] Dohán | Hydrocortisone and purinergic signaling stimulate sodium/iodide symporter (NIS)-mediated iodide transport in breast cancer cells[END_REF][START_REF] Tanosaki | Effect of ligands of nuclear hormone receptors on sodium/iodide symporter expression and activity in breast cancer cells[END_REF] and in animal models [START_REF] Kogai | Systemic retinoic acid treatment induces sodium/iodide symporter expression and radioiodide uptake in mouse breast cancer models[END_REF]. Even though these findings suggest their potential clinical use, as yet no clinical trials have been presently designed to this aim and additional strategies can be foreseen.

Epigenetic modifications, such as histone deacetylation, have been considered possible causes of NIS silencing in cancer cells. Our data clearly demonstrate that the deacetylase-inhibitor LBH589 induced NIS, both as mRNA and as a protein, and determined a significant iodide uptake in breast cancer cell lines. Furthermore, the observation that LBH589 strongly activates the transcription of the construct luc-NIS that we had transfected in breast cancer cells both ER-positive (MCF-7 and T47D) and -negative (MDA-MB 231) is consistent with a direct effect of the drug on NIS promoter. Our data suggest that DAC-inhibitor treatment could therefore modify the acetylation state of NIS promoter allowing its transcription and expression as already reported for other DAC-inhibitors [START_REF] Botrugno | Histone deacetylase inhibitors as a new weapon in the arsenal of differentiation therapies of cancer[END_REF]. The effect was evident regardless of ER status, even if both ER-positive MCF-7 and T47D cells are more sensitive than ER-negative MDA-MB 231 cells (the increase in NIS expression in ER-positive cells was about three times higher than in ER-negative cells). Thus, our study is the first report concerning the possibility to induce not only NIS mRNA expression but also iodide accumulation in breast cancer cells by using the pan-DAC inhibitor, LBH589.

In conclusion, the pan-DAC inhibitor LBH589 is a powerful anti-tumour agent and is able to induce NIS and iodide accumulation in breast cancer cells. The effects of LBH589 are independent from estrogen-sensitivity and ER -status of cells, making the drug of valuable use for diagnostic and therapeutic application in both estrogen-sensitive and -insensitive tumours.

LEGEND TO FIGURES

Figure 1 -

 1 Figure 1 -LBH589 effect on histone acetylation. Acetylation of histone H4 in MCF-7,

Figure 2 -

 2 Figure 2 -LBH589 effect of cell viability. Breast cancer cells were incubated with LBH589

Figure 3 -

 3 Figure 3 -LBH589 effect on apoptosis and cell cycle. Left Panels: ELISA detection of

Figure 4 -

 4 Figure 4 -LBH589 effect on NIS mRNA expression and NIS promoter activity. Left

Figure 5 -Table 1 .

 51 Figure 5 -LBH589 effects on NIS protein expression and iodide uptake. Upper Panels:

ACKNOWLEDGMENTS

We thank Novartis Pharma AG, Basel, Switzerland for providing us LBH589; Giuseppe Damante, Dipartimento di Scienze e Tecnologie Biomediche, Policlinico Universitario di Udine, Udine, Italy, for providing us with the luc-NIS construct; Daniela Taverna and Alessandra Solero, Dipartimento di Scienze Oncologiche, Università di Torino and M. B. C., Torino, Italy, for helping us with luciferase experiments. This study was supported by the Project "Alfieri 2007", Fondazione CRT, Torino, the Special Project "Oncology", Compagnia San Paolo, Torino, by MIUR and by Regione Piemonte to Giuseppe Boccuzzi


Henri-Hugues Fliche

Roland Triay
email: triay@cpt.univ-mrs.fr

Λ effect in the cosmological expansion of void

Keywords: cosmic web, cosmological parameters from LSS, cosmological simulations, cosmological perturbation theory

We investigate the dynamical effect of the cosmological constant Λ on a single spherical vacuum void evolving in the universe within a global solution of Newton-Friedmann models. As a result, the main characteristic is that the void expands with a huge initial burst up to match asymptotically the Hubble flow. The size of voids increases with Ω • and with Λ, which is interpreted as respectively by the gravitational attraction of borders from outside regions and by the gravitational repulsion of vacuum from the inner region. The Λ-effect on the kinematics intervenes significantly by amplifying the expansion rate at redshift z ∼ 1.7 for a background density parameter Ω • ∼ 0.3. For a class of parameters values, which corresponds in GR to spatially closed Friedmann models, it is interesting to note that a test particle in the inner region moves toward the border. Such a peculiar feature shows that the empty regions are swept out ; which stands as a stability criterion.

Introduction

Foam like patterns with large empty regions are present in the distribution of observed galaxies at scales up to 100 Mpc (see e.g., [32,[START_REF] Joevee | The Large Scale Structure of the Universe[END_REF]16,[START_REF] Pj | Proc. of Ninth Marcel Grossmann Meeting on General Relativity[END_REF]28,[START_REF] Croton | The 2dFGRS team[END_REF]14,[START_REF] Conroy | [END_REF]). Hence, one may wonder whether the voids would be the most suitable structures to be studied for improving our understanding on the formation process of large cosmological structures. Such a question has motivated investigations which have been performed -on their statistical properties, by improving identification techniques (see e.g., [27]), by exploring their formation process in a ΛCDM model through N-body simulations (see e.g., [13,31,12,22,[START_REF] Colberg | Outskirts of Galaxy Clusters : intense life in the suburbs[END_REF][START_REF] Van De Weygaert | Outskirts of Galaxy Clusters : intense life in the suburbs[END_REF]26]), and by probing their origins (see e.g., [24]); -on the kinematics of giant voids (see e.g., [17]) ; -and on their dynamics, by testing models of void formation (see e.g., [1,11,2]).

Herein, we propose to obtain a global solution describing the Newtonian dynamics of a spherical void in an expanding universe. The relevance of such a study in the context of cosmological structures can be justified as follows. From a theoretical point of view, it is clear that the dynamics of a single void does not allow to understand the evolution of a foam like structure but it is required as a first step of the investigate, however. From a standpoint of modeling observations, it appears that we can consider a homogeneous distribution of dust in which the void evolves. Indeed, at larger scales, according to observations, the dynamic of the cosmological expansion can be described by the Friedmann-Lemaître (FL) model, despite the presence of large scale structures. The reason can be found on theoretical grounds, due to the fact that the recession of galaxies together with the black body spectrum of Cosmological Background Radiation ensure that FL model provides us with a suitable description of the universe [START_REF] Souriau | Mécanique Statistique, Groupe de Lie et Cosmologie, in Géométrie symplectique et physique mathématique[END_REF].

Furthermore, the stability of FL models with respect to linear perturbations [18] enable us to use perturbative approaches in order to understand the large scale structures formation process. Such investigations are generally performed with numerical simulations based on a FL cosmological background in which the structures are embedded. However, since voids are not linear structures [1], a better understanding of the underlying dynamics requires a more adapted approach. With this in mind and the aim of understanding the effect of a cosmological constant Λ on the evolution of voids, we investigate the dynamics of a single empty spherical shell embeded in an uniformly distribution of dust which evolves with the cosmological expansion.

Our model is based on solutions of Euler-Poisson equations system for whom kinematics satisfy Hubble (cosmological) law. Except for the description of the gravitational waves, FL models can be described within such a framework (see e.g. [23]) and Newtonian treatment of perturbations leads to similar results as those obtained in general relativity (GR) [START_REF] Fliche | Évaluation des paramètres cosmologiques à l'aide des propriété optiques des quasars. Fluctuations des modèles de Friedmann-Lemaître[END_REF]. Analogous problems have been investigated in GR [29,19,20,30,21] but with a different aim with respect to the cosmological constant effect on the dynamics of the void (empty bubble).

Euler-Poisson equations system

The motion of a pressureless medium (dust) is described by its specific density ρ = ρ( r, t) and velocity v = v( r, t) at position r and time t. These fields are constrained by Euler-Poisson-Newton (EPN) equations system: i.e. Euler equations system

1 ∂ρ ∂ t + div (ρ v) = 0 (2.1) d v dt = ∂ v ∂ t + ∂ v ∂ r v = g (2.2)
where g = g( r, t) stands for the gravitational field, and they satisfy the modified Poisson-Newton equations system

-→ rot g = 0 (2.3) div g = -4πGρ + Λ (2.4)
where G is Newton constant of gravitation. The sign of the cosmological constant is chosen (Λ ≥ 0) such that the vacuum gravitational effect is repulsive. Accordingly to the status of gravitational field (which is not a vector field), we have chosen a cartesian coordinates system where g = 0 at its origin, see A.

In order to disentangle the evolution of structures from the cosmological expansion, it is convenient to write these equations with new coordinates, herein named reference coordinates. They are defined as follows

(t, x = r a ), a = a(t) > 0, ȧ(t) ≥ 0, a • = a(t • ) = 1 (2.5)
where a is a solution of Friedmann type differential equation

Ḣ + H 2 - Λ 3 + 4πG 3 ρ • a -3 = 0, H = ȧ a (2.6)
and ρ • stands for an arbitrary constant; herein, the dotted variables stand for time derivatives. This differential equation admits a integration constant

K • = 8πG 3 ρ • + Λ 3 -H 2 • , H • = H(a • ) (2.7)
The integration of Eq. (2.6) provides us with Friedmann equation

H 2 = Λ 3 - K • a 2 + 8πG 3 ρ • a 3 ≥ 0, H ∞ = lim a→∞ H = Λ 3 (2.8)
where H ∞ is the asymptotical value of the function H(a) when Λ > 0.

The differential equation given in Eq. (2.8) is integrated by a quadrature, and the function a(t) is obtained as a reciprocal mapping. It depends on three parameters chosen among Λ, ρ • , H • and K • , see Eq. (2.7). At this stage, it is important to mention that, excepted Λ, they do not identify (yet) with the cosmological parameters of FL model but they intervene solely in the choice of coordinates system. EPN equations system Eq. (2.1-2.4), reads in term of reference coordinates as follows

∂ρ c ∂ t + div (ρ c v c ) = 0 (2.9) ∂ v c ∂ t + ∂ v c ∂ x v c + 2H v c = g c (2.10) -→ rot g c = 0 (2.11) div g c = - 4πG a 3 (ρ c -ρ • ) (2.12)
where "div" and "

-→
rot" denote the differential operators with respect to variable x,

ρ c = ρa 3 , v c = d x dt (2.13)
act as the density and the velocity fields of the medium in the reference frame, and

g c = g a + 4πG 3a 3 ρ • - Λ 3 x (2.14)
as the acceleration field.

Newton-Friedmann and Vacuum models

In the reference frame, a uniform distribution of dust and an empty space are described, as obvious solutions of Eq. (2.9,2.10,2.11,2.12), respectively by :

1. a Newton-Friedmann model (NF), which is defined by

ρ c = ρ • , v c = 0, g c = 0 (2.15)
Herein, according to Eq. (2.8,2.13), a and H recover their usual interpretations as expansion parameter and Hubble parameter respectively, ρ • = ρa 3 identifies to the density of sources in a comoving space (of scalar curvature K • ). We focus our investigation to cosmological expansions which excludes bouncing solutions, i.e., ȧ ≥ 0.

Hence, an analysis on roots of third degree polynomials shows that the condition

K 3 • < (4πGρ • ) 2 Λ (2.16)
must be fulfilled. From primordial epochs, H decreases with time to reach its asymptotic value H ∞ . However, the kinematics has two distinct behaviors, characterized by the sign of K

• : if K • ≤ 0 then H decreases monotonically, but if K • > 0 then it
reaches first a minimum H m from where it grows to reach H ∞ from downward. This minimum is defined by

H m = H ∞ 1 - K 3 • Λ (4πGρ • ) 2 < H ∞ (2.17) at a = 4πGρ • K -1
• , it characterizes a loitering period. Let us remind that FL models are characterized by the following dimensionless parameters

λ • = Λ 3H 2 • , Ω • = 8πGρ • 3H 2 • , k • = K • H 2 • = λ • + Ω • -1 (2.18)
These notations are preferred to the usual Ω Λ = λ • and Ω K = -k • for avoiding ambiguities on the interpretation of cosmological parameters, see e.g., [START_REF] Fliche | [END_REF], [START_REF] Fliche | [END_REF]. A dimensional analysis of Eq. (2.7) shows that the Newtonian interpretation of k • corresponds to a dimensionless binding energy for the universe during its expansion; the lower k • the faster the cosmological expansion. In the FL world model, k • stands for the dimensionless spatial curvature of the comoving space; k • > 0 characterizes a spatially closed universe.

2. a Vacuum model (V), which is defined by

ρ c = 0, v c = (H ∞ -H) x, g c = 4πG a 3 ρ • x (2.19)

Spherical voids in Newton-Friedman universe

For avoiding misunderstandings in the interpretation of the dynamics, we use a covariant formulation of Euler-Poisson equations system (see A). The model of a spherical void embedded in a uniform distribution of dust is obtained by sticking together the V and NF models, both as local solutions of EPN equations system defined in Sec. 2.1. It accounts for the dynamics of their common border (i.e. the boundaries conditions), which is assumed to be a shell of null thickness.

Dynamical model

We consider three distinct media : a spherical thin shell (S) on which dust is uniformly distributed, an empty space (V) inside, and a uniform distribution of dust (NF) outside. For convenience in writing, the symbols S, V and NF denote respectively the related medium and the corresponding model. They behave such that S makes the juncture of V with NF according to Eq. (2.15,2.19). The tension-stress on S is assumed to be negligible, which is characterised by a (symmetric contravariant) stress-energy tensor defined as follows

T 00 S = (ρ S ) c , T 0j S = (ρ S ) c v j c , T jk S = (ρ S ) c v j c v k c (3.1)
The background is described by the following stress-energy tensor

T 00 NF = ρ c , T 0j NF = 0, T jk NF = 0 (3.2)
Because the eulerian functional

T (x → γ) = T µν S γ µν dtdS + T µν NF γ µν dtdV (3.3)
vanishes when γ reads in the form γ µν = 1 2 ∂µ ξ ν + ∂ν ξ µ , one has

S ∂ 0 ξ 0 + g j c ξ j + (∂ j ξ 0 + ∂ 0 ξ j -2Hξ j ) v j c + v j c v k c ∂ j ξ k (ρ S ) c dtx 2 dΩ = - NF ρ c ∂ 0 ξ 0 dtx 2 dxdΩ (3.4)
where dΩ stands for the solid angle element (see A). The radial symmetry of solutions enables us to write the reduced peculiar velocity and acceleration of a test particle located on the shell as follows

v c = α x, g c = β x (3.5)
where the functions α = α(t) and β = β(t) must be determined. A by part integration of Eq. ( 3.4) provides us with

t 2 t 1 (∂ 0 (ρ S ) c + 3(ρ S ) c α -ρ c αx) x 2 ξ 0 dt (3.6) = t 2 t 1 ∂ 0 ((ρ S ) c α) + 4(ρ S ) c α 2 + 2H(ρ S ) c α -(ρ S ) c βx -1 x 3 ξdt
where x = x stands for the radius of S and ξ = ξ 2 1 + ξ 2 2 + ξ 2 2 . This equality must be fulfilled for all bounded time intervals and compact support 1-forms. Hence, we easily derive the conservation equations for the mass

∂ 0 (ρ S ) c + (3(ρ S ) c -ρ c x) α = 0 (3.7)
and for the momentum

dα dt + 1 + ρ c (ρ S ) c x α 2 + 2Hα + β x = 0 (3.8)
With Eq. (2.5), the calculation of the gravitational force from the entire shell acting on a particular point 2 provides us with

β = 4πG a 3 ρ c 3 - (ρ S ) c 2x (3.9)
It is noticeable that (ρ S ) c = ρ c x/3 ensures that the mass forming the shell comes from its interior 3 . It must be understood as a particular solution of Eq. (3.7) which is compatible with initial conditions provided by FL model. By focusing our investigation to such a model, Eq. (3.8) transforms

dα dt + 4α 2 + 2Hα - 2πG 3
ρ c a 3 = 0 (3.10) With the dimensionless variable χ = 4 α H • a 2 (3.11)
where the ratio α H -1

• stands for the expansion rate of S in the reference frame, Eq. (3.10) tranforms into a Riccati equation

dχ da = Ω • - χ 2 a 1 P (a) (3.12)
where

P (a) = λ • a 4 -k • a 2 + Ω • a, P (1) = 1 (3.13)
According to Eq. (2.13,3.11), one has

x = x i exp a a i χda 4a P (a) (3.14)
where x i = x(t i ) and a i = a(t i ) stands for initial values (at time t = t i ). 2 The modified newtonian gravitation field reads g = Λ 3 -Gm r 3 r 3 This property agrees with spherical voids originating from a uniformly distributed matter, although other behaviors can be envisaged. Indeed, Eq. (3.7) reads ∂0δ + 3αδ = 0 where δ = (ρS)c -

1 3 ρcx = f (xi) exp -3 t t i
αdt accounts for the excess/defauft of the shell density with respect to that formed from an uniform distribution of matter, f (xi) stands for its initial value at t = ti.

Qualitative analysis

A meaningful qualitative analysis of the expansion of the void must be performed by means of dimensionless quantities. Herein, we chose the magnification X and the expansion rate Y , which are defined as follows

X = x x i , Y = α H • (3.15)
Their diagrams versus the expansion parameter a characterize adequately the expansion dynamics of S; they are obtained from Eq. (3.12, 3.14) by numerical integration. They can be easily translated in term of cosmic time t since a(t) is a monotonic function of t in the present investigation. The initial conditions lie on the expansion rate Y i and the formation date t i , as expressed by means of a i = a(t i ). Herein, we investigate voids that initially expand with Hubble flow Y i = 0 at a i = 0.003. Instead of having an exhaustive analysis with respect to cosmological parameters, we limit our investigation around the generally accepted values λ • = 0.7 and Ω • = 0.3. The kinematics of the expansion is discussed in Sec. 3.2.1, the dependence on cosmological parameters (herein named Ω-effect and Λ-effect respectively) in Sec. 3.2.2, the role of initial conditions in Sec. 3.2.3, and the results are synthesised in Sec. 3.2.4.

Kinematics

The expansion velocity of the shell S with respect to its centre reads

v = yH r, y = 1 + Y h , h = H H • (3.16)
where y stands for the corrective factor to Hubble expansion. According to Fig. 1, which shows y versus a for Ω • = 0.3 and for three values of λ • ∈ {0, 0.7, 1.4}, S expands faster than Hubble expansion (i.e., y > 1) all along its evolution. It starts with a huge increase of the velocity at early stages (such as a burst) with no significant dependence on Λ, and then it decreases for reaching asymptotically the Hubble flow 4 . The Λ effect is to delay the decreasing of the expansion velocity of S. On the diagram, it is characterised by a protuberance on the curves at z ∼ 1.7 for λ • > 0.7 (i.e., k • > 0) , the larger the λ • (i.e., k • ) the higher the bump. Such a feature is due to the existence of a minimum value H m of Hubble parameter H, which is reached during the cosmological expansion and is related to a coasting period, see Eq. (2.17). With respect to observation of distant voids, the Doppler shift between the edge and the center of a spherical void which is observed at redshift z does not exceed

∆ z = 1 (1 + z) v c c = XY 1 + z x i H • c (3.17)
where c is the speed of the light.

Dichotomy between cosmological parameters

The growth of spherical voids is investigated by means of X and Y diagrams with respect to cosmological parameters Ω • and λ • , with the aim of disentangling their related effects.

1. Ω-effect. -The dependence on the outward density is analyzed with a vanishing cosmological constant λ • = 0 and three values for the density parameter Ω • ∈ {0.15, 0.3, 0.45}. The comparison of diagrams in Fig. 2 tell us that the larger the Ω • the larger the magnification X. Such an effect results from the attraction of shell burst which freezes asymptotically up to matching Hubble expansion (Y = 0). Moreover, the larger the Ω • the larger the expansion rate. This trend is not significantly modified by other acceptable values of λ • .

2. Λ-effect. -The dependence on the cosmological constant is analyzed at constant Ω • = 0.3 and λ • ∈ {0, 0.7, 1.4}. The comparison of diagrams in Fig. 4 shows that the larger the λ • the larger the magnification X, which increases nonlinearly with λ • . evolution, but it has a cumulative effect which is reflected on X, see Fig. 1.

According to Eq. (2.7), the dependence of the dynamics of S on the parameter k • can be retrieved from above analysis; the higher k • the faster the S expansion, and a similar effect for the magnification. Let us point out an interesting feature that characterizes the sign of k • which appears on the motion in the reference frame of a test particle inside S. According to Eq. (2.19) :

• if k • ≤ 0 then the test particle moves toward the centre of the void;

• if k • > then it starts moving toward its border at date t = t ⋆ defined such that a(t ⋆ ) = 8 3 πGρ • K -1 • , which happens when Hubble expansion H < H ∞ . This feature, of sweeping out the inner part of S toward its border, stands for a stability criterion for voids which acts only for a spatially closed universe.

Dependence on initial conditions

-Figure 6 shows three curves corresponding to formation dates a i ∈ {0.003, 0.03, 0.3} by assuming Ω • = 0.3, λ • = 0.7 and with Y i = 0. As expected, the earlier the date of birth the larger the magnification X.

Figure 7 suggests the existence of a limiting curve a → Y defined by a i = 0, which characterises Ω • . The expansion rate curves related to other formation dates are located in lower part and reach it asymptotically. One can expect that the effects on the dynamics of growth resulting from reasonable initial expansion rates Y i = 0, related to physical processes (e.g., supernovae explosions [25]), are negligible at primordial epoch 5 , what legitimises the initial condition Y i = 0 at a i = 0.003. Evolutions with other initial conditions on Y i can be deduced from that since any given point (a i , Y i ) in the diagram belongs to a single evolution curve.

Synthesis

-As a result, the expansion of a spherical empty region does not show a linear regime, but it starts with a huge burst which freezes asymptotically up to match Hubble expansion. Its radius (in the reference frame) x increases from an initial size x i with the cosmological parameters Ω • and λ • . The related individual effects can be interpreted respectively by the gravitational attraction from the outer parts and by the gravitational repulsion of its borders (or equivalently, the vacuum). The larger these parameters the higher the magnification X = x/x i . The dynamics is sensitive to Ω-effect at primordial epochs and to Λ-effect later on. The evolution of its expansion velocity with time in the reference frame v c = Y H • x does not characterise λ • but Ω • . The cosmological constant intervenes mainly by delaying the decrease of the velocity expansion. The expansion rate evolutions related to formation dates show a common envelope curve which characterises the density parameter Ω • .

Discussion

It must be kept in mind that, based on two exact solutions of Euler-Poisson equations system, this model allows one to investigate properly the behaviour of a single spherical void. The connection conditions between the inner and the outer regions, which are investigated in a (covariant) Classical Mechanics, provides us unambiguously with the dynamics of the shell. Such a schema does not correspond to an embedding of a void in a cosmological background solution (as usual), but it stands for a non linear approach of NF models. However, while it can be useful in N-bodies simulations to improve voids dynamics, it requires stringent properties on the space distribution of neighboring matter and ignores GR effects. At this step, one expects that it can apply solely to spherical voids with reasonable size in order to guarantee reliable results. With the aim of using the characteristics of voids to determine the values of the cosmological parameters, it is interesting to mention that the property of K • of being an integration constant (see Eq. 2.7, 2.18) enables us to use k • to classify the dynamics of the cosmological expansion. With this in mind, let us note that the spatially closed world models k • > 0 are characterized by a significant expansion of voids which reaches its maximum at redshift z ∼ 1.7 (with Ω • ∼ 0.3), the larger the λ • (i.e., k • ) the higher the expansion rate. The inner part of voids shows a de Sitter expansion v = Λ/3 r which sweeps out it, what can be interpreted as a stability criterion.

Conclusion

Using Euler-Poisson equations system with cosmological constant, we investigate the dynamics of an isotropic universe constituted by a spherical (empty) void surrounded by a uniform distribution of dust. It turns out that the behaviours of both regions (inside and outside the shell) coincide with Newton-Friedmann (NF) solutions, which ensures a consistent model. The dynamics of the void expansion is analyzed in the reference (comoving) frame

The general behaviour of the void expansion shows a huge initial burst, which freezes asymptotically up to match Hubble expansion. While the corrective factor to Hubble law on the shell depends weakly on the cosmological constant Λ at early stages, it enables us to disentangle significantly cosmological models around redshift z ∼ 1.7. The magnification of spherical voids increases with the cosmological parameters related to the density and to the cosmological constant. In case of spatially closed Friedmann models, the empty regions are swept out, which provides us with a stability criterion. It could be applied to observational data, as long as the matter distribution in the outer region of the single void is reasonably uniform

A. Covariant formulation of Euler-Poisson equations system

Souriau's covariant formulation of Euler-Poisson equations [33] can be summarized as follows. The geometrical interpretation of Newton dynamics [6] shows that the component of gravitational field g can be identified to the unique non vanishing Christoffel components of newtonian connexion Γ j 00 = -g j Hence, one obtains their expression in the new coordinates system defined in Eq. (2.5), and the only non null component reads (Γ c ) j k0 = Hδ j k , (Γ c ) j 00 = -g j c

Let T be a functional6 defined on the set of symmetric density tensors x → γ on newtonian spacetime R 4

T (x → γ) = R 4
T µν γ µν dtdV where dtdV stands for the volume element and T for a symmetric contravariant tensors which accounts for the media. The measure density T is eulerian if and only if it vanishes for all covariant tensor fields that reads

γ µν = 1 2 ∂ξ ν ∂x µ + ∂ξ µ ∂x ν
where ∂ stands for the covariant derivative7 and x → ξ for a compact support 1-form. In such a case, it is obvious to show that ∂T µν ∂x µ = 0 can be interpreted as the Euler equations, in any coordinates systems.

Figure 1 :

 1 Figure 1: The corrective factor to Hubble expansion.

Figure 2 :Figure 3 :

 23 Figure 2: The magnification X -dependence on density.

Figure 4 :Figure 5 :

 45 Figure 4: The magnification X -dependence on the cosmological constant.

Figure 6 :Figure 7 :

 67 Figure 6: The magnification X -dependence on the formation date.

Note that in the second Euler equation, we have ∂ v ∂ r i j = ∂v i ∂r j .

Although the present epoch (a = 1) appears quite peculiar because of the relative proximity of these curves, this turns out to be solely an artefact since they (will) cross at a > 1 but not in a single point.

Since Hubble expansion is all the more important towards the past, the earlier the formation date the weaker this effect, according to Eq.(3.16).

A functional is a real-valued function on a vector space, usually of functions. It is often used in the variational calculus, in particular in Mechanics.

Let us remind the definition of the covariant derivative ∂ξν ∂x µ = ∂ξν ∂x µ + Γ λ µν ξ λ


B Cocquelin

G Lucas-Leclin
email: gaelle.lucas-leclin@institutoptique.fr

D Holleville

N Dimarcq

I Sagnes

M Myara

A Garnache

P Georges

COMPACT AND ROBUST SINGLE-FREQUENCY DIODE-PUMPED VECSEL AT THE CESIUM D 2 LINE FOR ATOMIC CLOCKS

This work reports on an optically-pumped vertical external-cavity surface-emitting laser emitting around 852 nm dedicated to atomic physics experiments with cold Cs atoms. The design of the semiconductor active structure has been optimized to provide a low threshold. A low-power diode-pumped compact prototype has been developed with improved stability. With this setup, we obtained a 17-mW single frequency emission exhibiting large tunability around the Cesium D 2 line. The laser linewidth has been measured to less than 500 kHz on a 10 ms time.

INTRODUCTION

In atomic physics experiments, complex laser benches are used to perform trapping, cooling, manipulation and optical detection of atoms [START_REF] Cheinet | Compact laser system for atom interferometry[END_REF]. Actually the different stages of these cold-atom experiments have distinct constraints on laser sources, such as high output power (> 200 mW) for efficient atom cooling or stimulated-Raman transitions, but narrow-linewidth emission (< 500 kHz) for optical detection. In any case, stable single-frequency operation and fine tunability over some GHz around the atomic transitions are requested. Currently, available laser sources show intrinsic limitations in fulfilling all the required properties. Consequently, several lasers are used simultaneously to provide different optical features, while a single laser source would greatly improve the compactness, the efficiency and the simplicity of these set-ups. Optically-pumped Vertical External-Cavity Surface-Emitting Lasers (VECSELs) combine the approach of diode-pumped solid-state lasers and engineered semiconductor lasers, generating both circular diffraction limited output beams and high powers [START_REF] Kuznetsov | Design and Characteristics of High-Power (>0.5 W CW) Diode-Pumped Vertical-External-Cavity Surface-Emitting Semiconductor Lasers with Circular TEM 00 Beams[END_REF]. These lasers benefit from the knowledge in semiconductor fabrication. The power scaling possibilities of these semiconductor thin disks to obtain multi-watts output powers have already been demonstrated [START_REF] Chilla | High power Optically Pumped Semiconductor Lasers[END_REF][START_REF] Lutgen | 8-W high-efficiency continuouswave semiconductor disk laser at 1000 nm[END_REF]. Furthermore the utilization of a resonant periodic gain design ensures a spatially and spectrally-homogeneous gain, which is favourable for single-frequency operation [START_REF] Abram | Narrow linewidth operation of a tunable optically pumped semiconductor laser[END_REF]. VECSELs appear then to be an interesting way to achieve compact and efficient tunable single-frequency sources for metrology and spectroscopy.

In this paper we present the design of a specific laser structure emitting near 852 nm -corresponding to the Cs D 2 line. In a very simple and compact laser cavity setup, we have obtained a single-frequency line tunable around the Cesium D 2 line. The frequency stabilization on an atomic line has been performed, and the laser linewidth has been characterized through a beat note with a reference laser.

DESIGN OF THE SEMICONDUCTOR ACTIVE STRUCTURE

The active region of the semiconductor structure has been specifically designed for laser emission around 852 nm.

It is optically-pumped in the quantum-well (QW) barriers, which prescribes a pump wavelength below 720 nm. Since red laser sources are neither as efficient nor as powerful in that spectral range than in the infrared, the active structure has been carefully designed with the aim to reach a low laser threshold while keeping an optical gain high enough to compensate for cavity losses. Seven QWs correspond to an optimum which minimizes the pump intensity at threshold for an output coupler which transmission T is about a few percents. Further increase of the QWs number would result in a higher threshold intensity.

DESCRIPTION OF THE LASER SOURCE

With the aim to develop a low-power single-frequency source dedicated to the of Cesium atoms, a compact monolithic external cavity with improved thermal and mechanical stability has been designed. We chose a single-transverse-mode laser diode as the pump source in order to limit intensity noise transfer from the pump to the laser emission. The pump was thus a Mitsubishi red laser diode delivering up to 150 mW at 658 nm for an operating current of 245 mA. The pump beam was focused on a 20 µm radius spot with a pair of aspheric lenses, at the incidence angle of 70° relatively to the structure, which circularizes the elliptic shape of the incident pump beam on the semiconductor chip. The external cavity was formed by a 12 mm-concave mirror R = 99 % at 852 nm mounted on a piezo-electric transducer (PZT) (see Fig. 2). The laser cavity length was carefully adjusted in order to optimize the overlap between the cavity mode and the pump one. The pump system, the semiconductor chip and the external mirror have been integrated on the same mount. The overall setup fitted within a 52×52×58 mm 3 cube. The temperature of the laser cavity was stabilized with a thermoelectric cooler, and the semiconductor device was thermally regulated with an independent controller. The whole set-up was protected by Dural® and acoustic isolant coatings to improve both its thermal and acoustic isolation. Then the laser remains running for weeks without needs for re-alignment. Without any spectrally selective element In this compact prototype, the laser output power is limited by the available pump power to 17 mW (Fig. 3) at an active structure temperature of 10° C. The threshold was reached for an incident pump power of 52 mW, and the slope efficiency was 17% (with regard to incident pump power) without any evidence of thermal roll-over up to the maximum 150-mW pump power. The emission was linearly polarized, and the beam was diffractionlimited with a quality factor M 2 < 1.1. The emitted wavelength shifted from 850.5 nm at threshold to 852.2 nm at maximum pump power. Without any frequency-selective intracavity element, we observed a stable single-frequency operation within a large range of operating conditions of the substrate temperature and pump current, with a side-mode suppression ratio higher than 25 dB (measurementlimited). This was checked with a high finesse (F=130) scanning Fabry Perot with a free-spectral range (FSR) of 37.5 GHz larger than the FSR of the laser cavity. This single-frequency operation is attributed to the nearlyideal gain homogeneity of VECSELs sources which has been extensively studied in [START_REF] Garnache | Single-Frequency operation of External-Cavity VCSELs: Non-linear multimode temporal dynamics and quantum limit[END_REF]. Actually the characteristic time needed for the laser spectrum to collapse to a single-mode is about 1 ms in our experimental conditions, faster than acoustic and thermal fluctuations in our laser set-up.

Pump Diode

Output coupler + PZT

3/4

Fig. 3 : Output power (full symbols, left axis) and wavelength (circles, right axis) of the VECSEL source with (green) and without (blue) intracavity etalon.

With an intracavity Fabry-Perot etalon

To force the laser wavelength independently of the operating conditions, we chose to insert a 26-µm thick uncoated silica etalon inside the cavity. The laser wavelength is then only controlled by the etalon orientation over 9 nm, and does not change with either the temperature (over ∆T = 30°C) or the available pump power. In the meantime the output power and the efficiency decreased respectively to 8 mW at the maximum available pump power and 12% with respect to the incident pump power; the threshold increased to 85 mW due to the extra losses introduced by the etalon. By tuning the external-cavity length with the piezoelectric ceramic, we achieved a continuous tunability over 15 GHz (0.03 nm) without any mode hop. This large continuous tunability of the laser frequency, owing to our short-length cavity, makes it possible to scan continuously the optical transitions of interest, distant from 9.192 GHz, corresponding to the hyperfine levels ( 62 S 1/2 , F = 3) and ( 62 S 1/2 , F = 4) of the 133 Csatom ground state (Fig. 4).

EVALUATION OF THE SPECTRAL PROPERTIES

With the etalon inside the cavity, the laser frequency has been locked at the side of a Doppler-free transition of the D 2 line, through a low-frequency (f < 2 kHz) 2integration-stage servo loop on the piezoelectric ceramic. The laser linewidth of our source has been evaluated through the beat note measurement between our OP-VECSEL and an extended-cavity diode laser (ECDL) which linewidth has been evaluated to 130 kHz at -3 dB from previous measurements [START_REF] Baillard | Interferencefilter-stabilized external-cavity diodes lasers[END_REF]. In order to suppress low-frequency drifts of the laser lines, the OP-VECSEL and the ECDL were locked to different sub-Doppler Cs lines. The heterodyne signal around F 0 = 135 MHz was detected by a fast photodiode, then amplified and measured with a RF analyzer. The full width at half maximum of the beat note is 500 kHz, which is mainly attributed to the OP-VECSEL (see Fig. 5). The FWHM of the Lorentzian fit of the spectrum wings ( ! " # " 0 > 1 MHz) is 70 kHz. These values are due to extra acoustic, thermal and electrical noises which are unavoidable under normal laboratory environment.

CONCLUSION AND OUTLOOK

From a low-power compact prototype we have obtained a pump-limited output power of 17 mW under singlefrequency operation on the Cesium D 2 transition. Our source was continuously tunable around the desired wavelength. Furthermore a beat-note experiment has established that the laser linewidth was about 500 kHz. These results demonstrate the potentialities of our source as an alternative in the optical benches of Cesium atomic clocks. Actually the VECSEL is already adequate for the detection of atoms in clock experiments. Recently, preliminary experiments towards higher output power have already resulted in a 120-mW singlefrequency emission of our VECSEL under 1-W diode pumping. The further increase of the laser power will require a reduction of the thermal resistance of the semiconductor chip in order to withstand high-power pumping. Standard solutions such as removing the GaAs substrate and soldering the structure onto a highconductivity substrate or a heatsink are under investigation.

Fig. 1 : 4 Distributed

 14 Fig.1 : Active structure design The semiconductor chip has been grown by metalorganic chemical-vapor deposition (MOCVD) on a 350µm thick (100) GaAs substrate. It contains a multilayer

Fig. 2

 2 Fig. 2 : Laser prototype 4. LASER OPERATION 4.1.Without any spectrally selective element In this compact prototype, the laser output power is limited by the available pump power to 17 mW (Fig.3) at an active structure temperature of 10° C. The threshold was reached for an incident pump power of 52 mW, and the slope efficiency was 17% (with regard to incident pump power) without any evidence of thermal roll-over up to the maximum 150-mW pump power. The emission was linearly polarized, and the beam was diffractionlimited with a quality factor M 2 < 1.1. The emitted wavelength shifted from 850.5 nm at threshold to 852.2 nm at maximum pump power. Without any frequency-selective intracavity element, we observed a stable single-frequency operation within a large range of operating conditions of the substrate temperature and pump current, with a side-mode suppression ratio higher than 25 dB (measurementlimited). This was checked with a high finesse (F=130) scanning Fabry Perot with a free-spectral range (FSR) of 37.5 GHz larger than the FSR of the laser cavity. This single-frequency operation is attributed to the nearlyideal gain homogeneity of VECSELs sources which has been extensively studied in[START_REF] Garnache | Single-Frequency operation of External-Cavity VCSELs: Non-linear multimode temporal dynamics and quantum limit[END_REF]. Actually the characteristic time needed for the laser spectrum to collapse to a single-mode is about 1 ms in our experimental conditions, faster than acoustic and thermal fluctuations in our laser set-up.

Fig. 4 :

 4 Fig. 4 : Continuous tunability of the laser frequency with PZT voltage; red curve : Cs absorption spectrum; green curve : transmission of a 1.5 GHz low-finesse etalon.

Acknowledgments: This work has been supported by the Délégation Générale à l'Armement (N°0534004), and the Agence Nationale de la Recherche (ANR-07-BLAN-0320-03). B. Cocquelin acknowledges the CNRS and CNES for the funding of his PhD.

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