Hany Onsy Habashy

Emad A Rakha

Mohammed Aleskandarany

Mohammed Ahmed

Andrew R Green

Ian O Ellis
email: ian.ellis@nottingham.ac.uk

Desmond G Powe

Prof O Ian

Ellis

FOXO3a nuclear localisation is associated with good prognosis in luminal-like breast cancer

Keywords: Breast carcinoma, FOXO3a, Oestrogen receptor, Prognosis, Immunohistochemistry, Luminal-like

Oestrogen receptor (ER)-positive breast cancer (BC) constitutes a heterogeneous group of tumours with regard to outcome and response to therapy. Accurate stratification of ER-positive BC according to risk of relapse and response to therapy will be achieved through an improved understanding of ER and ER-related biological pathways. Recent studies have identified FOXO3a (Forkhead box O3a) transcription factor as an intracellular mediator of ERα expression and as an important downstream target of the Akt/PI3K pathway indicating a biological and potential clinical role for FOXO3a in ER-positive BC.

In this study, we investigated the clinical relevance and biological associations of FOXO3a protein expression, using tissue microarrays and immunohistochemistry, in a large series of patients with invasive breast cancer. FOXO3a protein expression showed both nuclear and/or cytoplasmic staining patterns. FOXO3a predominant nuclear expression was positively associated with biomarkers of good prognosis including PgR, FOXA1, and p27 expression. There was an inverse association with mitotic counts, MIB1 growth fraction, C-MYC and PIK3CA expression. With respect to patient outcome, FOXO3a nuclear localisation was associated with longer BC specific survival (p<0.001) and longer distant metastasis free interval (p=0.001), independently of the well-established breast cancer prognostic factors.

In conclusion, our results demonstrate the biological and prognostic role of FOXO3a protein expression and its subcellular localization in ER-positive/luminal-like BC possibly through its involvement in controlling cell proliferation.

Introduction

Oestrogen receptor (ER) positive tumours comprise the majority of breast cancers, accounting for 60-70% of cases and are generally expected to show good response to hormonal treatment and favourable clinical outcome [START_REF] Murphy | Steroid receptors in human breast tumorigenesis and breast cancer progression[END_REF]. However, ER-positive tumours are heterogeneous with respect to their clinical behaviour and biology. By studying novel biomarkers with strong association with ER signalling it should be possible to gain a better understanding of the biology of ER-positive disease.

FOXO3a (FKHRL1) gene belongs to the forkhead family of transcription factors [START_REF] Yang | A New Fork for Clinical Application: Targeting Forkhead Transcription Factors in Cancer[END_REF] and their activity is regulated by several post-translational modifications, including phosphorylation and acetylation [START_REF] Tsai | Crystal structure of the human FOXO3a-DBD/DNA complex suggests the effects of post-translational modification[END_REF]. Zou et al 2008 [START_REF] Zou | Forkhead box transcription factor FOXO3a suppresses estrogendependent breast cancer cell proliferation and tumorigenesis[END_REF] have reported that FOXO3a can suppress ERα-dependent breast cancer cell proliferation and tumourigenesis in the MCF-7 breast cancer cell line, suggesting a crosstalk between FOXO3a and ER signalling pathways in ER dependent breast cancer [START_REF] Zou | Forkhead box transcription factor FOXO3a suppresses estrogendependent breast cancer cell proliferation and tumorigenesis[END_REF]. Other studies demonstrated FOXO3a has an important intracellular mediator role in ERα expression, suggesting possible therapeutic intervention [START_REF] Guo | Forkhead Box Transcription Factor FOXO3a Regulates Estrogen Receptor Alpha Expression and Is Repressed by the Her-2/neu/Phosphatidylinositol 3-Kinase/Akt Signaling Pathway[END_REF]. Importantly, FOXO3a is a downstream target in the Akt/PI3K pathway and when phosphorylated, is prevented from translocating to the nucleus resulting in its loss of functional activity. In contrast, FOXO3a dephosphorylation leads to nuclear localisation and subsequent target gene activation [START_REF] Brunet | Akt Promotes Cell Survival by Phosphorylating and Inhibiting a Forkhead Transcription Factor[END_REF][START_REF] Vara | PI3K/Akt signalling pathway and cancer[END_REF][START_REF] Huang | Dynamic FoxO transcription factors[END_REF][START_REF] Yang | ERK promotes tumorigenesis by inhibiting FOXO3a via MDM2-mediated degradation[END_REF][START_REF] Burgering | Decisions on life and death: FOXO Forkhead transcription factors are in command when PKB/Akt is off duty[END_REF]. Therefore, as a target within the Akt/PI3K signalling pathway FOXO3a regulates the expression of proapoptotic genes, cell cycle-regulated genes, and genes that control cellular homeostasis [START_REF] Brunet | Akt Promotes Cell Survival by Phosphorylating and Inhibiting a Forkhead Transcription Factor[END_REF][START_REF] Burgering | Cell cycle and death control: long live Forkheads[END_REF]. Alvarez el al [START_REF] Alvarez | Forkhead transcription factors contribute to execution of the mitotic programme in mammals[END_REF] suggest that an efficient mitotic programme depends on downregulation of Akt/PI3K and consequent induction of FOXO3a transcriptional activity. However, there is also evidence that an Akt-independent mechanism of FOXO3a regulation exists. In vitro co-transfection of FOXO3a and IKK resulted in strong inhibition of FOXO3a activity independent of Akt [START_REF] Hu | I[kappa]B Kinase Promotes Tumorigenesis through Inhibition of Forkhead FOXO3a[END_REF].

In breast cancer, FOXO3a activity has been shown to elevate p27 resulting in cell cycle arrest [START_REF] Accili | FoxOs at the Crossroads of Cellular Metabolism, Differentiation, and Transformation[END_REF]. Furthermore, nuclear FOXO3a can induce cellular apoptosis through upregulation of Fas ligand (Fas-L) [START_REF] Brunet | Akt Promotes Cell Survival by Phosphorylating and Inhibiting a Forkhead Transcription Factor[END_REF] and Bim [START_REF] Burgering | Cell cycle and death control: long live Forkheads[END_REF][START_REF] Stahl | The Forkhead Transcription Factor FoxO Regulates Transcription of p27Kip1 and Bim in Response to IL-2[END_REF] and has been implicated in resistance to oxidative stress and longevity [START_REF] Kops | Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress[END_REF]. Other studies have highlighted the importance of FOXO3a for maintenance of the hematopoietic stem cell pool [START_REF] Miyamoto | Foxo3a Is Essential for Maintenance of the Hematopoietic Stem Cell Pool[END_REF][START_REF] Tothova | FoxOs Are Critical Mediators of Hematopoietic Stem Cell Resistance to Physiologic Oxidative Stress[END_REF].

It has been reported that activation of FOXO3a could induce p53-dependent apoptosis even in cells with a transcriptionally inactive p53 [START_REF] You | Regulation of transactivation-independent proapoptotic activity of p53 by FOXO3a[END_REF].

FOXO3a may have therapeutic implications because some clinical anticancer treatments target FOXO3a through three main oncogenic kinases (Akt, IKK, and ERK) [START_REF] Yang | A New Fork for Clinical Application: Targeting Forkhead Transcription Factors in Cancer[END_REF][START_REF] Myatt | The emerging roles of forkhead box (Fox) proteins in cancer[END_REF][START_REF] Yang | A New Fork for Clinical Application: Targeting Forkhead Transcription Factors in Cancer[END_REF]. For instance, nuclear localization of FOXO3a could potentially improve the effectiveness of anti-EGFR agents such as gefitinib by mediating proliferative arrest [START_REF] Krol | The transcription factor FOXO3a is a crucial cellular target of gefitinib (Iressa) in breast cancer cells[END_REF]. Gefitinib treatment causes cell cycle arrest and induces apoptosis due to the effects of FOXO3a dephosphorylation and nuclear translocation at Akt sites. In contrast, resistant cells show phosphorylated FOXO3a are restricted to the cytoplasm [START_REF] Krol | The transcription factor FOXO3a is a crucial cellular target of gefitinib (Iressa) in breast cancer cells[END_REF]. Furthermore, up-regulation of FOXO3a by paclitaxel has been reported to increase Bim mRNA and protein level with subsequent induction of apoptosis in breast cancer cells [START_REF] Sunters | FoxO3a Transcriptional Regulation of Bim Controls Apoptosis in Paclitaxeltreated Breast Cancer Cell Lines[END_REF][START_REF] Sunters | Paclitaxel-Induced Nuclear Translocation of FOXO3a in Breast Cancer Cells Is Mediated by c-Jun NH2-Terminal Kinase and Akt[END_REF].

The value of FOXO3a as a prognostic biomarker for ER-positive luminal-like breast cancer remains unclear. Therefore, in this study, we have investigated the clinical relevance and biological associations of FOXO3a protein expression in a large series of patients with luminal-like ER-positive invasive breast cancers using highthroughput tissue microarrays (TMAs) and immunohistochemistry.

Materials and Methods

Patient selection and tissue microarray construction

This study was approved by the Nottingham Research Ethics Committee 2 under the title "Development of a molecular genetics classification of breast cancer".

Formalin fixed paraffin embedded (FFPE) tissue microarrays (TMAs) were prepared from a series cases of primary operable (stage I and II) breast carcinoma cases from patients age <70 presenting consecutively to the Nottingham Breast Unit with tumours of less than 5 cm diameter between 1989 and 1998 as previously reported [START_REF] El-Rehim | High-throughput protein expression analysis using tissue microarray technology of a large well-characterised series identifies biologically distinct classes of breast cancer confirming recent cDNA expression analyses[END_REF]. This consecutive patient series is well characterized and contains patients' clinical and pathological data including patients' age, histologic tumour type, primary tumour size, lymph node status, mitotic count and histologic grade [START_REF] Elston | Pathological Prognostic Factors in Breast-Cancer .1. the Value of Histological Grade in Breast-Cancer -Experience from a Large Study with Long-Term Follow-Up[END_REF], Nottingham prognostic index (NPI) [START_REF] Galea | The Nottingham Prognostic Index in Primary Breast-Cancer[END_REF], vascular invasion (VI), therapy and follow-up data. In addition, data on a large panel of biomarkers with strong relevance to breast cancer were available including oestrogen receptor-α (ER), ER-related genes (progesterone receptor (PgR), androgen receptor (AR), FHIT, FOXA1, CARM1, and PELP1), proliferation, apoptosis and cell cycle-related genes (BCL2, p27, p53, C-MYC, and MIB1), and biomarkers related to PI3K pathway (PIK3CA) [START_REF] El-Rehim | High-throughput protein expression analysis using tissue microarray technology of a large well-characterised series identifies biologically distinct classes of breast cancer confirming recent cDNA expression analyses[END_REF][START_REF] Habashy | Forkhead-box A1 (FOXA1) expression in breast cancer and its prognostic significance[END_REF][START_REF] El-Rehim | Expression of luminal and basal cytokeratins in human breast carcinoma[END_REF].

Patient management was based on the Nottingham Prognostic Index (NPI) score and ER status as previously described [START_REF] Habashy | Forkhead-box A1 (FOXA1) expression in breast cancer and its prognostic significance[END_REF][START_REF] Rakha | Invasive lobular carcinoma of the breast: Response to hormonal therapy and outcomes[END_REF], patients within the good prognostic NPI group (≤3.4) did not receive adjuvant systemic therapy. Hormonal therapy (Tamoxifen ± Zoladex if premenopausal) was given to patients with ER-positive tumours and NPI scores of >3.4. Pre-menopausal patients with moderate and poor prognostic NPI groups were given chemotherapy (Cyclophosphamide, Methotrexate, and 5-Flourouracil). ER-positive postmenopausal patients with moderate or poor NPI were offered Hormonal therapy, while ER negative patients received CMF if fit to receive these cytotoxic agents with no concurrent disease that were considered as potential contraindication to the use of chemotherapy.

Data are maintained on prospective basis for breast cancer specific survival (BCSS), development of distant metastases (DM) and/or locoregional tumour recurrence. BCSS was defined as the time (in months) from the date of the primary surgical treatment to the time of death from breast cancer. Distant metastasis free interval DMFI was defined as the interval (in months) from the date of the primary surgical treatment to the date of development of the first distant metastasis. FOXO3a expression was assessed in the whole patient series (n=934), and a cohort of ERpositive / luminal-like patients (n=633).

Immunohistochemistry

Rabbit polyclonal antibody to FOXO3a (Antibody 9467, Cell Signalling Technology, Danvers, MA) was optimized at a working dilution of 1:50 using FFPE TMA sections and full-face sections of breast cancer tissue to assess the staining distribution.

Immunohistochemical optimization and staining of FOXO3a was performed using a DakoCytomation Techmate 500 plus (DakoCytomation, Cambridge, UK) immunostainer with a linked streptavidin biotin (LSAB) technique in accordance with the manufacturer's instructions after microwave antigen retrieval in 0.01M citrated buffer pH6 and as previously described [START_REF] El-Rehim | High-throughput protein expression analysis using tissue microarray technology of a large well-characterised series identifies biologically distinct classes of breast cancer confirming recent cDNA expression analyses[END_REF]. Negative controls were performed by omitting the primary antibody and substitution with a diluent. Sections were counterstained in haematoxylin and coverslipped using DPX mounting medium.

Western blotting was performed on breast cancer cell lysates of the human breast cancer cell line MCF-7 to confirm the specificity of the FOXO3a antibody used in immunohistochemistry. MCF-7 cells were obtained from the American Type Culture Collection (Rockville, MD, USA) and cultured in RPMI 1640 medium in T75 flasks supplemented with 10% foetal calf serum (FCS), penicillin (100 IU/ml) and streptomycin (100 μg/ ml). The sub-confluent cells were washed with PBS, then 30μl of protease inhibitors (Sigma Aldrich) were added to 470μl of ice-cold lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% TritonX-100, 0.5% sodium deoxycholate, 1 mM EDTA, 0.1% SDS). Western blotting was done on the cell lysates to confirm the specificity of the antibody used in immunohistochemistry. Lysates (20µg) were added to 4X SDS loading buffer with 5% β-mercaptoethanol (Sigma Aldrich, UK) and denatured by heating at 100ºC for 10 minutes prior to loading then added for 5 minutes into ice. Samples were subjected to Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) using a 10% resolving polyacrylamide gel and transferred onto a Hybond-P PVDF membrane (Amersham Bioscience, Buckinghamshire, UK). After blocking with 5% milk powder 0.1% TPBS (Tween20 in PBS solution) for 60 minutes, the membrane was then incubated with 1:1000 dilution of the FoxO3a rabbit polyclonal Antibody (9467) at 4°C overnight.

The membrane was washed with 0.1% PBS/Tween20 3 times for 5 minutes each then incubated for 1 h at room temperature with a horseradish peroxidase-linked secondary antibody (Sigma Aldrich) (1: 4000, anti-rabbit) diluted with 5% milk powder PBS containing 0.1% Tween20. After a further 3 washes, the membrane was visualized enhanced chemiluminescence reagents (Amersham Bioscience, Buckinghamshire, UK). The monoclonal Anti-β-actin antibody (Sigma Aldrich) in a dilution of 1:2000 against the ubiquitous β-actin protein was used.

Assessment of IHC staining results

Staining was initially evaluated on full face sections to assess the expression and the distribution of staining. Firstly, FOXO3a TMAs IHC staining results were categorised into negative and positive expression, regardless of FOXO3a localisation.

Examination of the TMAs has shown that some cases showed nuclear pattern and others were mainly cytoplasmic. Since the expression pattern and localisation of FOXO3a protein expression show variable biological functions, we categorised the positive cases according to whether they showed predominant nuclear (N) or predominant cytoplasmic (C) localisation. Both patterns scored separately using the percentage of the positive cells in each TMA core. Cases were categorised as showing a nuclear or cytoplasmic pattern in 50% or more of the informative TMA core provided that there is more than 20 % variation between both patterns. We have defined the cases with obvious overlap (less than 20 % variation, n=31) and were excluded to ensure a clear separation in their patterns of expression. The cases were scored without the knowledge of patient outcome.

The H-score (histochemical score) was used to assess the intensity of staining and the percentage of stained cells for FOXA1 [START_REF] Habashy | Forkhead-box A1 (FOXA1) expression in breast cancer and its prognostic significance[END_REF], PELP1 [START_REF] Habashy | The prognostic significance of PELP1 expression in invasive breast cancer with emphasis on the ER-positive luminal-like subtype[END_REF], CARM1, and PIK3CA [START_REF] Aleskandarany | PIK3CA expression in invasive breast cancer: a biomarker of poor prognosis[END_REF].

The H-score (Histochemical score) was used to assess staining intensity and percentage of stained tumour cells following immunohistochemistry. Staining intensity was scored 0, 1, 2 or 3 (negative to strong) and the percentage of positive cells at each intensity subjectively estimated to produce a final score in the range 0-300. For other biomarkers we used the percentage of the positive cells. Intensity of C-MYC staining was scored as negative, low, moderate or strong. MIB1 staining was done on full face breast cancer sections. Suppliers, dilutions, antigen retrieval methods, cutoffs, and methods of staining assessment used for the biomarkers included in this study is summarised in (Table 1).

Statistical analysis

We have reported our results according to the REMARK criteria for reporting tumour marker prognostic studies [START_REF] Mcshane | Reporting recommendations for tumor marker prognostic studies[END_REF][START_REF] Mcshane | REporting recommendations for tumor MARKer prognostic studies (REMARK)[END_REF]. Statistical analysis was performed using SPSS 16 statistical software (SPSS Inc., Chicago, IL, USA). Association between FOXO3a staining patterns and different clinicopathological parameters was evaluated using Chi-square test or Fishers exact test. Survival analysis was estimated by the Kaplan-Meier plots and Log Rank test to assess significance. Multivariate Cox proportional hazard regression models were used to evaluate any independent prognostic effect of the variables with 95% confidence interval. A p-value of <0.05 was considered to reflect a significance.

Results

The median age of the patients was 56 years (range 27-70). At the time of the primary diagnosis, Forty seven percent (47%) of patients had tumours less than 2 cm in size and 31.5% had grade 2 tumours. During follow-up, 30.6% of the patients developed metastatic disease.

The specificity of the FOXO3a antibody was confirmed using Western blotting (Fig

Correlation of FOXO3a protein expression and other clinicopathological variables

The tumour-specific FOXO3a IHC staining characteristics were initially categorised into negative and positive expression (showing either nuclear or cytoplasmic expression), regardless of FOXO3a localisation. FOXO3a expression status did not show significant associations with the other clinicopathological variables including tumour grade, size, stage, NPI and vascular invasion (p>0.05).

Subsequently, the cases were categorised according to the pattern of expression into nuclear and non-nuclear, the latter including cytoplasmic predominant localisation and negative expression. Analysis of whole patient series revealed that FOXO3a nuclear localisation is positively associated with low mitotic counts, lower grade tumour, less frequent development of distant metastasis (p<0.001) and tumour recurrence (p=0.001) (Table 2). Its expression was significantly associated with markers of good prognosis (Table 3).

In the luminal-like cohort (ER cutoff 10%), FOXO3a nuclear expression pattern was associated with low mitotic counts (p=0.008), and less frequent development of distant metastasis (p<0.001) and tumour recurrence (p=0.035) (Table 4). The nuclear pattern showed significant positive associations with molecular biomarkers associated with good prognosis including PgR (p=0.004), FHIT (p=0.008), FOXA1 (p<0.001) and p27 (p=0.004) expression. It also showed an inverse correlation with the expression of cell proliferation-related markers, MIB1 expression (p=0.039) and with PIK3CA (p=0.006) (Table 5).

Patients' outcome

In whole patient series, initial univariate analysis of FOXO3a expression status (as positive versus negative) was not associated with BCSS (Log Rank (LR) =0.005,

Multivariate analyses

Since many potential prognostic factors may interact with specific therapies and therefore are compounded by the effect of adjuvant hormone therapy and chemotherapy, we have included the systemic therapy groups (given versus not given) in the multivariate analysis together with the other well established prognostic variables such as MIB1, PgR, tumour size, stage, grade to assess the prognostic independence of nuclear FOXO3a expression in the ER+ patient cohort.

FOXO3a nuclear expression was an independent prognostic factor for predicting better outcome in terms of longer BCSS (Hazard ratio (HR)=0.392, p=0.006, 95%CI=0.202-0.760)(Table 6) and longer DMFI (HR=0.530, p=0.020, 95%CI=0.310-0.906) (Table 7) in ER-positive / luminal-like breast cancer.

Discussion

Oestrogen receptor (ER) plays an important role in breast cancer progression and response to therapy and is typically expressed in the most frequent biological class of breast cancers. However ER-positive breast cancer does not appear to be a homogenous group; some tumours respond to therapy and others do not. There is therefore a requirement for improved understanding of the biology of ER-positive breast cancer which could be achieved through studying ER-related pathways and their downstream targets. One of the important pathways that has been implicated in the pathogenesis of the poor prognosis ER-positive (luminal B) subtype is the Akt/PI3K pathway and its related genes [START_REF] Loi | Molecular analysis of hormone receptor positive (luminal) breast cancers -What have we learnt?[END_REF]. FOXO3a is a key downstream target in this pathway which prompted us to study its expression and associations in breast cancer particularly in ER-positive / luminal-like subtype.

The Akt/PI3K pathway regulates the sub-cellular localization of FOXO3a by phosphorylation which prevents the protein from translocating to the nucleus to regulate transcription [START_REF] Brunet | Akt Promotes Cell Survival by Phosphorylating and Inhibiting a Forkhead Transcription Factor[END_REF]. This indicates that absence of nuclear FOXO3a expression in a tumour, with either complete absence or cytoplasmic localisation, should indicate phosphorylation by Akt. This may represent an important biological mechanism responsible in part for poor prognosis in ER-positive breast cancer, thus removing a constraint to cellular proliferation and potentially to tumourigenesis through an active Akt/PI3K pathway. This proposal is supported by our finding that absence of nuclear expression of FOXO3a is associated with poorer outcome and shows a significant association with PIK3CA, a marker strongly related to Akt function. Other breast cancer studies have also shown an association between Akt/PI3K activation and cytoplasmic FOXO3a expression pattern with decreased patient survival, in agreement with our findings [START_REF] Hu | I[kappa]B Kinase Promotes Tumorigenesis through Inhibition of Forkhead FOXO3a[END_REF].

In this study we did not find a significant association with survival when patients were categorised into either negative or positive FOXO3a expressers per se. Instead, we found that subcellular localisation indicates functional relevance as evidenced here by more favourable outcome in patients with predominant nuclear expression. Supporting these findings, previous studies have shown that nuclear FOXO3a induces the expression of genes that inhibit cell cycle progression such as the CDK inhibitors [START_REF] Zou | Forkhead box transcription factor FOXO3a suppresses estrogendependent breast cancer cell proliferation and tumorigenesis[END_REF][START_REF] Brunet | Akt Promotes Cell Survival by Phosphorylating and Inhibiting a Forkhead Transcription Factor[END_REF]. Subsequently, we found a significant positive association between nuclear FOXO3a and the expression of the cell cycle inhibitor p27 implying a role in the induction of cell cycle arrest.

In this patient series including ER-positive / luminal-like subtype, nuclear localisation of FOXO3a was associated with markers of good prognosis such as FHIT, PgR [START_REF] Bardou | Progesterone Receptor Status Significantly Improves Outcome Prediction Over Estrogen Receptor Status Alone for Adjuvant Endocrine Therapy in Two Large Breast Cancer Databases[END_REF], and FOXA1 expression which is required for the expression of 50% of ER-regulated genes [START_REF] Thorat | FOXA1 expression in breast cancer is associated with Luminal subtype and good prognosis[END_REF]. Furthermore, we have also shown that nuclear FOXO3a expression is significantly associated with longer BCSS and DMFI which implies its role in stratification of ER-positive groups into prognostic subgroups, possibly explained by a tumour suppressor function associated with cell cycle arrest.

Previous studies have shown that loss of FOXO3a function indicated by its absence or by cytoplasmic localisation is positively associated with markers of increased proliferation [START_REF] Accili | FoxOs at the Crossroads of Cellular Metabolism, Differentiation, and Transformation[END_REF]. We have found that BC, especially luminal-like cases, expressing nuclear FOXO3a are characterised by low proliferation, specifically low mitotic frequency and low MIB1 expression. In addition, we found an inverse association between FOXO3a nuclear expression and C-MYC protein supporting the proposal that the Akt/PI3K/FOXO pathway modulates MYC function by inhibition of MYC-dependent transcription resulting in decreased cellular proliferation [START_REF] Delpuech | Induction of Mxi1-SR{alpha} by FOXO3a Contributes to Repression of Myc-Dependent Gene Expression[END_REF]. Taken together, our findings support the interaction of FOXO3a as a downstream target of Akt/PI3K pathway with markers related to proliferation and cell cycle, a role which is independent of the systemic therapy as shown here by our multivariate analysis results.

In conclusion, our results demonstrate the biological and prognostic role of FOXO3a protein expression, as a downstream target of AKT pathway, and its subcellular localization in BC. Loss of nuclear translocation of FOXO3a could tilt the balance in favour of increased proliferation and more aggressive behaviour in luminal-like breast cancers. Nuclear expression was significantly associated with improved DMFI in comparison to cytoplasmic pattern.

  1). In normal breast tissue, FOXO3a expression was detected mainly in the nuclei of mammary epithelial cells (Fig2a). In the malignant tissues, FOXO3a showed both nuclear and cytoplasmic staining patterns but one pattern were obviously dominant.In whole patient, about 23 % showed predominant nuclear expression pattern and 34 % cytoplasmic pattern while 3 % showed both with less 20% difference in the predominant pattern. In ER-positive patient cohort, 26 % showed nuclear expression pattern (Fig 2b) and 31 showed % cytoplasmic pattern (Fig 2c).

  p=0.942) nor DMFI (LR =0.015, p=0.904) but when the localisation of expression was considered FOXO3a nuclear expression was associated with better outcome in terms of longer BCSS (LR=24.079, p<0.001) and longer DMFI (LR =15.996, p<0.001). In the luminal-like ER-positive cohort of patient (n=633), (median follow up time =126 months), univariate analysis of survival showed no associations between FOXO3a expression status (as positive versus negative) and patient outcome in terms of BCSS (LR =0.234, p=0.628) (Fig 3a) or DMFI (LR=0.198, p=0.656). However, FOXO3a nuclear localisation showed a significant association with both longer BCSS (LR =15.813, p<0.001) (Fig 3b) and longer DMFI (LR=11.836, p=0.001) (Fig 3c).When we analysed patient survival using categorisation of the cohort into three groups: predominant nuclear, predominant cytoplasmic and negative, our results showed that subcellular localisation differences of FOXO3a are associated with striking survival differences. Specifically there is a contrast between nuclear and cytoplasmic expression localisation where nuclear pattern showed the most favourable BCSS (LR =18.279, p<0.001) (Fig 3e) and longer DMFI (LR=14.775, p=0.001) (Fig 3f) in ER+ luminal-like cancer. According to systemic therapy groups When systemic therapy was considered, similar associations of longer survival were found in the subgroup of ER-positive patients who did not receive adjuvant systemic therapy (n=222) with regards DMFI (LR=10.110, p= 0.001) (Fig 3d) and in the subgroup of patients who received tamoxifen monotherapy (n=221) with regards BCSS (Log Rank (LR) =5.201, p=0.023).

Figures legendsFigure 1 :

 1 Figures legends

Figure 2a :

 2a Figure 2a: FOXO3a expression in normal tissue (mainly nuclear).

Figure 2b :

 2b Figure 2b: Predominant FOXO3a nuclear expression in low grade ductal cancer.

Figure 2c :

 2c Figure 2c: Predominant FOXO3a cytoplasmic expression in high grade ductal cancer.

Figure 3a :

 3a Figure 3a: Kaplan Meier plot of FOXO3a expression status regardless of its

Figure 3b :

 3b Figure 3b: Kaplan Meier plot of FOXO3a nuclear verse non-nuclear protein

Figure 3c :

 3c Figure 3c: Kaplan Meier plot of FOXO3a nuclear expression with respect of DMFI

Figure 3e :

 3e Figure 3e: Kaplan Meier plot of FOXO3a expression patterns with respect of BCSS.

Figure 3f :

 3f Figure 3f: Kaplan Meier plot of FOXO3a expression patterns with respect of DMFI.

Table 1 :

 1 Suppliers, dilutions, antigen retrieval methods, cutoffs, and methods of staining assessment used for the biomarkers included in this study

	^=Scored as (0, absent; 1, weak; 2, moderate; 3, strong expression)
	*=H score

Table 2 :

 2 Associations between FOXO3a immunostaining patterns and various clinicopathological parameters in the whole series

	Variable	Non-Nuclear localisation	localisation Predominant Nuclear	Total	χ 2	p-value
	Age
	<40	59(85.5)	10(14.5)	69
	40-50 51-60	215(78.2) 235(79.2)	60(21.8) 61(20.6)	275 295	5.276	0.153
	>60	194(73.8)	69(26.6)	263
	Tumour size
	≤2 cm >2 cm	335(78.1) 367(77.6)	94(21.9) 106(22.4)	429 473	0.032	0.873
	Lymph node stage
	1(Negative)	413(76.6)	126(23.4)	539
	2(1-3 LN)	219(79.1)	58(20.9)	277	1.169	0.557
	3(>3 LN)	69(81)	16(19)	84
	Tumour grade
	1	97(71.3)	39(28.7)	136
	2	214(74.6)	73(25.4)	287	9.115	0.010
	3	391(81.6)	88(18.4)	479
	Vascular invasion
	No/Probable Definite	457(77.9) 244(77.7)	130(22.10 70(22.3)	587 314	0.003	1.000
	NPI
	Good	164(73.5)	59(26.5)	223
	Moderate	408(78.3)	113(21.7)	521	4.364	0.113
	Poor	131(82.4)	28(17.6)	159
	Mitotic counts
	1	204(73.6)	73(26.4)	277
	2	109(67.7)	52(32.2)	161	20.308 <0.001
	3	365(83.5)	72(16.5)	437
	DM
	No Positive	451(73.5) 242(87.4)	163(26.5) 35(12.6)	614 277	21.375 <0.001
	Recurrence
	No Positive	374(73.9) 314(82.8)	132(26.1) 65(17.2)	506 379	10.000	0.002

Table 3 :

 3 Associations between FOXO3a immunostaining patterns and other biomarkers in the whole series

					χ 2
	Variable	Non-Nuclear	Predominant Nuclear	Total		p-value
		localisation	localisation
		N (%)	N (%)
	ER
	Negative	202(83.8)	39(16.20	241	6.356	0.013
	Positive	466(75.9)	148(24.1)	614
	PgR
	Negative	319(84.4)	59(15.6)	378	17.014	<0.001
	Positive	344(72.6)	130(27.4)	474
	AR
	Negative	257(82.5)	55(17.5)	312	4.364	0.035
	Positive	378(75.9)	120(24.1)	498
	FOXA1
	Negative	319(85.1)	56(14.9)	375	15.145	<0.001
	Positive	234(73.1)	86(26.9)	320
	CARM1
	Negative/low	159(80.7)	38(19.30)	197	1.806	0.405
	Moderate	281(81.2)	65(18.8)	346
	Strong	97(75.8)	31(24.20	128
	PELP1
	Negative/low	76(74.5)	26(25.5)	102	2.612	0.271
	Moderated	412(80.7)	99(19.3)	511
	Strong	69(75.8)	22(24.2)	91
	p53
	Negative	474(77.8)	135(22.2)	609	0.080	0.856
	Positive	196(78.8)	53(21.3)	249
	MIB1
	Low	126(71.2)	51(28.8)	177	5.180	0.028
	High	407(79.5)	105(20.5)	512
	PIK3CA
	Negative	175(70.9)	72(29.1)	247	11.224	0.001
	Positive	444(81.5)	101(18.5)	545
	p27
	Negative	318(83.9)	61(16.1)	379	11.229	0.001
	Positive	272(73.9)	96(26.1)	368
	C-MYC
	Negative	87(88.8)	11(11.2)	98
	Low	203(80.6)	49(19.4)	252	8.113	0.044
	Moderate	203(75.5)	66(24.5)	269
	Strong	113(80.10	28(19.9)	141
	FHIT
	Negative	118(88.7)	15(11.3)	133	11.092	0.001
	Positive	475(75.5)	154(24.5)	629
	BCL2
	Negative	236(80.8)	56(19.2)	292	1.896	0.193
	Positive	312(76.5)	96(23.5)	408

Table 4 :

 4 Associations between FOXO3a immunostaining patterns and various clinicopathological parameters in the luminal-like cohort

	Variable		Predominant
		Non-Nuclear
			Nuclear	Total		p-
		localisation			χ 2
			localisation			value
		N (%)
			N (%)
	Age
	<40	24(72.7)	9(27.3)	33
	40-50 51-60	135(73.8) 159(78.7)	48(26.2) 43(21.3)	183 202	1.535	0.674
	>60	147(75.4)	48(24.6)	195
	Tumour size
	≤2 cm >2 cm	231(75.2) 234(76.5)	76(24.8) 72(23.5)	307 306	0.126	0.777
	Lymph node stage
	1(Negative)	269(73.9)	95(26.1)	364
	2(1-3 LN)	153(77.7)	44(22.3)	197	2.135	0.344
	3(>3 LN)	41(82)	9(18)	50
	Tumour grade
	1	88(71.5)	35(28.5)	123
	2	185(73.5)	67(26.5)	253	4.905	0.086
	3	191(80.6)	46(19.34)	237
	Vascular invasion
	No/Probable Definite	302(76.1) 162(75.3)	95(23.9) 53(24.70	397 215	0.040	0.844
	NPI
	Good	142(72.4)	54(27.6)	196
	Moderate	247(75.5)	80(24.5)	327	5.078	0.079
	Poor	77(84.6)	14(15.40	91
	Mitotic counts
	1	184(73.3)	67(26.7)	251
	2	90(68.2)	42(31.8)	132	9.643	0.008
	3	172(82.3)	37(17.7)	209
	DM
	No Positive	306(71.5) 155(85.6)	122(28.5) 26(14.4)	428 181	13.825 <0.001
	Recurrence
	No Positive	251(72.3) 207(79.9)	96(27.7) 52(20.1)	347 259	4.627	0.035

Table 5 :

 5 Associations between FOXO3a immunostaining patterns and other biomarkers in the luminal-like cohort

	Variable		Predominant	Total	χ 2	p-value
		Non-Nuclear
			Nuclear
		localisation
			localisation
		N (%)
			N (%)
	PgR
	Negative	123(84.2)	23(15.8)	146	7.968	0.004
	Positive	333(72.7)	125(27.3)	458
	AR
	Negative	110(79.1)	29(20.9)	140	0.860	0.423
	Positive	326(75.3)	60(24.7)	433
	FOXA1
	Negative	183(84.7)	33(15.3)	216	14.177 <0.001
	Positive	180(70)	77(30)	257
	CARM1
	Negative/low	121(78.1)	34(21.9)	155	2.128	0.345
	Moderate	191(79.6)	49(20.4)	240
	Strong	44(71)	18(29)	62
	PELP1
	Negative/low	57(74)	20(26)	77	2.136	0.344
	Moderated	278(79)	74(21)	352
	Strong	40(71.4)	16(28.6)	56
	p53
	Negative	374(76.8)	113(23.2)	487	0.0366	0.545
	Positive	86(74.1)	30(25.9)	116
	MIB1
	Low	102(68.9)	46(31.1)	148	4.580	0.039
	High	253(78.1)	71(21.9)	324
	PIK3CA
	Negative	136(69.4)	60(30.6)	196	8.120	0.006
	Positive	276(80.2)	68(19.8)	344
	p27
	Negative	162(83.9)	31(16.1)	193	8.438	0.004
	Positive	221(72.7)	83(27.3)	304
	C-MYC
	Negative	56(88.9)	7(11.1)	63
	Low	137(78.3)	38(21.7)	175	6.756	0.080
	Moderate	138(73.4)	50(26.60	188
	Strong	69(79.3)	18(20.7)	87
	FHIT
	Negative	64(87.7)	9(12.3)	73	6.573	0.008
	Positive	353(73.8)	125(26.2)	478
	BCL2
	Negative	92(78.6)	25(21.40	117	0.351	0.616
	Positive	275(76)	87(24)	362

Table 6 :

 6 Cox model of predictors of BCSS in the luminal-like breast cancer

		P value	HR	95% CI
	Variable			Lower	Upper
	FOXO3a nuclear localisation	0.006	0.392	0.202	0.760
	PgR expression	0.049	0.642	0.413	0.997
	MIB1 expression	0.011	2.105	1.184	3.742
	Tumour size	0.001	2.228	1.411	3.520
	LN stage	0.000	1.746	1.290	2.363
	Tumour grade	0.006	1.629	1.152	2.302
	Endocrine therapy	0.502	0.846	0.519	1.379
	Chemotherapy	0.305	0.715	0.377	1.358

Table 7 :

 7 Cox model of predictors of DM in the luminal-like breast cancer

	Table legends

Table 1 :

 1 Suppliers, dilutions, antigen retrieval methods, cutoffs, and methods of staining assessment used for the biomarkers included in this study

Table 2 :

 2 Associations between FOXO3a immunostaining patterns and various clinicopathological parameters in the whole series

Table 3 :

 3 Associations between FOXO3a immunostaining patterns and other biomarkers in the whole series

Table 4 :

 4 Associations between FOXO3a immunostaining patterns and various clinicopathological parameters in the luminal-like cohort

Table 5 :

 5 Associations between FOXO3a immunostaining patterns and other biomarkers in the luminal-like cohort

Table 6 :

 6 Cox model of predictors of BCSS in the luminal-like patient cohort

Table 7 :

 7 Cox model of predictors of DM in the luminal-like patient cohort

Acknowledgement

We thank the ministry of higher education (Egypt) for funding HO Habashy, M Ahmad, M Aleskandarany and E Rakha and Breast Cancer Campaign for funding A Green.

All authors declare no conflict of interest.


Paola Maroni

Emanuela Matteucci

Alessandro Luzzati

Giuseppe Perrucchini

Paola Bendinelli

Dr Maria Alfonsina Desiderio
email: a.desiderio@unimi.it

Nuclear co-localization and functional interaction of COX-2 and HIF-1α characterize bone metastasis of human breast carcinoma

Keywords: bone microenvironment, bone metastasis, breast cancer, COX-2, HIF-1

Introduction

Metastasis formation is the worst clinical complication of neoplastic disease, and is the primary cause of mortality in breast carcinoma patients [START_REF] Psaila | The metastatic niche: adapting the foreign soil[END_REF]. Notwithstanding the clinical relevance of the metastatic process, the molecular basis of growth advantage, tropism for the secondary site (bones, lungs), and osteolysis are partly known.

We have generated a pre-clinical model of human-osteolytic metastasis using 1833-high bone metastatic clone [START_REF] Previdi | Interaction between human-breast cancer metastasis and bone microenvironment through activated hepatocyte growth factor/Met and -catenin/Wnt pathways[END_REF], suitable to study microenvironment involvement in breast-cancer metastasis outgrowth and progression. The 1833-metastasis is characterized by a partial reversion to an epithelial phenotype, compared to the frankly mesenchymal phenotype of the parental MDA-MB231 cells. HIF-1 and NF-kB transcription factors drive this revertant phenotype, and are controlled at epigenetic level [START_REF] Bendinelli | NF-kB activation, dependent on acetylation/deacetylation, contributes to HIF-1 activity and migration of bone metastatic breast carcinoma cells[END_REF].

The current most recognized theory for metastasis engraftment is the metastatic niche model, presuming that the tumor cell does not solely dictate its own fate but that the formation of a hospitable microenvironment is essential-not just permissive-to enable a disseminating tumor cell to spawn a secondary tumor growth [START_REF] Chiodoni | Matricellular proteins: from homeostasis to inflammation, cancer and metastasis[END_REF]. These niches form as a result of factors secreted by tumor and stroma cells, and can either be newly induced or be adaptations of pre-existing physiological niches, such as stem cell niches of hematopoietic organs [START_REF] Psaila | The metastatic niche: adapting the foreign soil[END_REF][START_REF] Joyce | Microenvironmental regulation of metastasis[END_REF]. Thus, the gene signature associated with poor prognosis facilitates the emergence of metastatic cells in the primary tumor, but the specific set of genes associated with bone metastasis seems responsible for the cellular activities necessary for metastatisation [START_REF] Kang | A multigenic program mediating breast cancer metastasis to bone[END_REF][START_REF] Weigelt | Breast cancer metastasis: markers and models[END_REF][START_REF] Klein | Identification of brain-and bone-specific breast cancer metastasis genes[END_REF]. For completeness, the metastatic capacity might be considered as a late, acquired event in tumorigenesis and during secondary growth, through epigenetic control of transcription [START_REF] Kopfstein | Metastasis: cell-autonomous mechanisms versus contributions by the tumor microenvironment[END_REF]10].

The aim of the present paper is to clarify some molecular mechanisms underlying bone metastasis formation by human-breast carcinoma, taking into account the plasticity of the metastatic phenotype through responsiveness to physical (hypoxia) and biological (hepatocyte growth factor, HGF, and transforming growth factor-1, TGF-1) stimuli [START_REF] Previdi | Interaction between human-breast cancer metastasis and bone microenvironment through activated hepatocyte growth factor/Met and -catenin/Wnt pathways[END_REF][START_REF] Kingsley | Molecular biology of bone metastasis[END_REF]. To this purpose, we studied specimens of human-bone metastasis of breast cancer and the 1833-xenograft model. The data were confirmed by in vitro experiments using 1833 clone, that permitted to stress the differences with parental MDA-MB231 cells. The functional impact on metastatic phenotype of microenvironmental stimuli was examined through the triggering of a network of transcription factors, and the expression of specific target genes. The rationale of the present study was that a complex array of stimuli forms the metastatic microenvironment, and that it might influence specific sets of genes and thus the metastatic phenotype and the organotropism. Gene expression depends, in fact, on the specificity of the stimulus and the interaction of transcription factors showing consensus sites on the gene promoter. Transcription factors do not work alone but in cooperation. The transcriptional properties of a particular factor are influenced either by its position, relative to other factors bound to a given promoter, or by the abundance of transcriptional cofactors in a given cell type in a certain context [START_REF] Fry | Context-dependent transcriptional regulation[END_REF]. Our study deals with the molecular mechanisms involved in cyclooxygenase-2 (COX-2) expression and function in bone metastasis of breast cancer. COX-2 promoter is characterized by consensus sites for various transcription factors [START_REF] Lee | Hepatocyte growth factor regulates cyclooxygenase-2 expression via -catenin, Akt, and p42/p44 MAPK in human bronchial epithelial cells[END_REF], but their functional role in bone metastatic process has never been investigated. Moreover, COX-2 intracellular localization might be implicated in the control of the activity of some transcription factors, such as HIF-1-the master regulator of oxygen homeostasis [START_REF] Semenza | HIF-1 and mechanisms of hypoxia sensing[END_REF]by controlling the expression and function of the inducible α subunit.

Here, the subcellular localization of COX-2 and the mechanisms responsible for the regulation of its expression/localization were evaluated, in relation to HIF-1α expression and HIF-1 activity, due to the possible importance for biology, prognosis, therapy and gene-expression pattern of metastasis. HIF-1α expression in carcinomas is regulated by HIF-1 and NF-kB activities, as demonstrated using Semenza's HIF-1α-promoter constructs [START_REF] Tacchini | Hepatocyte growth factor signalling stimulates hypoxia inducible factor-1 (HIF-1) activity in HepG2 hepatoma cells[END_REF]. We give for the first time a fairly comprehensive view of the pattern of HIF-1-target genes, under the influence of metastasismicroenvironmental stimuli and after blockade of COX-2 activity or TGF-1-typeI receptor.

In animal models obtained with mouse (TM40D-MB or 4T1.2) and human (MDA-MB231) cells, COX-2/PGE2 system participates in osteolysis [START_REF] Kang | A multigenic program mediating breast cancer metastasis to bone[END_REF][START_REF] Hiraga | Stimulation of cyclooxygenase-2 expression by bone-derived transforming growth factor-beta enhances bone metastases in breast cancer[END_REF][START_REF] Singh | COX-2 involvement in breast cancer metastasis to bone[END_REF][START_REF] Li | Increased COX2 expression enhances tumor-induced osteoclastic lesions in breast cancer bone metastasis[END_REF] but the distinct functions contributing to breast-carcinoma metastasis remain largely unknown. HGF/Met couple is important for the cross-talk between metastasis and microenvironment [START_REF] Previdi | Interaction between human-breast cancer metastasis and bone microenvironment through activated hepatocyte growth factor/Met and -catenin/Wnt pathways[END_REF], and its activation by hypoxia might be hypothesized based on data from normal and neoplastic cell types [START_REF] Pennacchietti | Hypoxia promotes invasive growth by transcriptional activation of the met protooncogene[END_REF][START_REF] Desiderio | Hepatocyte growth factor in invasive growth of carcinomas[END_REF]. CXCR4 may drive tumor cells to the secondary sites (lymph nodes, bones) where CXCL12 specific ligand is produced [START_REF] Balkwill | Cancer and the chemokine network[END_REF], but the experimental data on human metastases are still preliminary [START_REF] Shim | Lower expression of CXCR4 in lymph node metastases than in primary breast cancers: potential regulation by liganddependent degradation and HIF-1α[END_REF][START_REF] Yoshitake | Expression of SDF-1α and nuclear CXCR4 predicts lymph node metastasis in colorectal cancer[END_REF]. Moreover, HIF-1α correlates with Met and metastasis in node-negative breast cancer [START_REF] Semenza | HIF-1 and tumor progression: pathophysiology and therapeutics[END_REF][START_REF] Chen | Hypoxia-inducible factor-1alpha correlates with MET and metastasis in node-negative breast cancer[END_REF]. HIF-1α protein is overexpressed in multiple types of human carcinomas, including primary-breast cancer (29%) as well as in lymph node metastases (69%) [START_REF] Zhong | Overexpression of hypoxia-inducible factor 1α in common human cancers and their metastases[END_REF].

The adaptability of bone-metastasis phenotype to hypoxic microenvironment at the bonesecondary growth site resulted in unique-molecular events, such as increased HIF-1α expression and nuclear localization both in the xenograft model and in the human specimens of bone metastasis, possibly orchestrated by COX-2. A fundamental permissive role was played by TGF-1 and HGF, produced by the metastatic cells in the hypoxic bone microenvironment.

Materials and methods

Reagents

Recombinant-human HGF and TGF-1, human HGF and TGF-1 immunoassays (ELISA) and anti-human CXCR4(MAB 172) antibody were from R&D System (Abigdon, United Kingdom).

Antibodies for human-Met(C12) and human-HGFα(C20), for VEGF(A20), HIF-1β(Arnt1-N19), vinculin and B23 were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-COX-2 antibody was from Cayman Chemical (Ann Arbor, MI). Anti-HIF-1α antibodies for immunohistochemistry (clone H1α67) and Western blot (clone54) were from Novus Biological (Littleton, CO) and BD-Transduction Laboratories (Franklin Lakes, NJ). Anti-cytokeratin(Pan) clone-AE1/AE3 was from Zymed Laboratories Inc (San Francisco, CA). Alexa Fluor488 and 568 antibodies were from Molecular Probes (Eugene, OR, USA). N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS398) and 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide hydrate (SB431542) were from Sigma-Aldrich (St Louis, MO). SN50 was from Alexis. CGP049090 was kindly given by Novartis (Basel, Switzerland).

Cells and Xenograft Model

Parental MDA-MB231 cells, the derived bone-metastatic clone 1833 wild-type and retrovirally transfected with triple-reporter construct (1833/TGL) were a generous gift of J. Massagué (Memorial Sloan-Kettering Cancer Center, New York). The xenograft model was obtained with 1833/TGL [START_REF] Previdi | Interaction between human-breast cancer metastasis and bone microenvironment through activated hepatocyte growth factor/Met and -catenin/Wnt pathways[END_REF]. MDA-231BO were kindly given by T. Yoneda (The University of Texas, San Antonio).

Surgical Specimens

Ten surgical specimens of bone metastasis, originating from breast carcinomas, were from patients who provided informed consent, in accordance with Declaration of Helsinki.

Histology and Immunohystochemistry

Serial sections of fixed and decalcified xenograft-model bone metastasis and human-bone metastasis specimens [START_REF] Previdi | Interaction between human-breast cancer metastasis and bone microenvironment through activated hepatocyte growth factor/Met and -catenin/Wnt pathways[END_REF], were probed with anti-COX-2 (1:50) and anti-HIF-1α (1:1000) overnight at 4° C.

Immunofluorescence

The 1833 cells (8 x 10 4 ) on coverslips were exposed to (i) 24-h hypoxia [START_REF] Tacchini | Hepatocyte growth factor signalling stimulates hypoxia inducible factor-1 (HIF-1) activity in HepG2 hepatoma cells[END_REF], in the presence or the absence of 1 M NS398 (COX-2 inhibitor) (4-h inhibitor pre-treatment and 20-h hypoxia plus the inhibitor) [START_REF] Hiraga | Stimulation of cyclooxygenase-2 expression by bone-derived transforming growth factor-beta enhances bone metastases in breast cancer[END_REF]; (ii) 100 g/ml cycloheximide for 2 or 4 h after 12-hypoxia exposure [START_REF] Matteucci | Nuclear localization of active HGF receptor Met in aggressive MDA-MB231 breast carcinoma cells[END_REF]; (iii) 10 M clasto-lactacystin--lactone (LLnL) 4 h before HGF-treatment (24 h) [START_REF] Tacchini | Hepatocyte growth factor signalling stimulates hypoxia inducible factor-1 (HIF-1) activity in HepG2 hepatoma cells[END_REF]. The antibodies used were: anti-COX-2 (1:30) and anti-HIF-1α (1:50). The images were collected at X400 magnification under Eclipse 80i, Nikon Fluorescence microscope.

Plasmids and Cell Transfection

The cells were transfected with the construct containing the multimer for Hypoxia-responsive element (6-HRE, pGL3PGK6TKp), NF-kB-binding sites (3-NFkB), TCF-binding sites (6-TCF, TOPFLASH), AP-1-binding sites (4-AP-1) from P.J. Ratcliffe, M. Hung, J. Gottardi and N.

Colburn [START_REF] Previdi | Interaction between human-breast cancer metastasis and bone microenvironment through activated hepatocyte growth factor/Met and -catenin/Wnt pathways[END_REF][START_REF] Bendinelli | NF-kB activation, dependent on acetylation/deacetylation, contributes to HIF-1 activity and migration of bone metastatic breast carcinoma cells[END_REF], or 5-Ets-1 consensus sequences [10]. The expression vector (200 ng) for HIF-1α or HIF-1β (L. Poellinger, Karolinska Institute, Stockholm, Sweden) was co-transfected with the genereporters. After overnight starvation (0.1% fetal bovine serum, FBS), 24-h treatment with 200 ng/ml HGF [START_REF] Previdi | Interaction between human-breast cancer metastasis and bone microenvironment through activated hepatocyte growth factor/Met and -catenin/Wnt pathways[END_REF] or 5 ng/ml TGF-1 [START_REF] Hiraga | Stimulation of cyclooxygenase-2 expression by bone-derived transforming growth factor-beta enhances bone metastases in breast cancer[END_REF], also under hypoxia, was performed. Cells exposed to hypoxia alone were not starved. COX-2-reporter plasmid (-1432/+59)Luc or (-327/+59)Luc (S. Wilkinson, Vanderbilt University, Nashville, TN) was co-transfected with expression vectors for the dominant negative of ARNT (corresponding to mutated HIF-1 subunit), Jun (TAM67) or Ets-1 (EBHHB) [START_REF] Bendinelli | NF-kB activation, dependent on acetylation/deacetylation, contributes to HIF-1 activity and migration of bone metastatic breast carcinoma cells[END_REF]10], or treated with 50 g/ml SN50 (NF-kB inhibitor) or 10 M CGP049090 (TCF-inhibitor) [START_REF] Lepourcelet | Smallmolecule antagonists of the oncogenic Tcf/-catenin protein complex[END_REF][START_REF] Yue | Tumor necrosis factor-alpha-mediated suppression of adipocyte apolipoprotein E gene transcription: primary role for the nuclear factor (NF)-kappaB pathway and NFkappaB p50[END_REF]. As internal control, Renilla luciferase plasmid was co-transfected, and Firefly/Renilla luciferase activity ratios were calculated by the software.

Western Blot Assay

Total extracts were prepared from cells exposed to 10 M LLnL 4 h before TGF-1, HGF or hypoxia treatment. Total and nuclear extracts were obtained from cells exposed to TGF-1 or hypoxia also in the presence of 1 M NS398 or 5 M SB431542. For Western blots, total (100 g) and nuclear (50/100 g) protein-extracts were used. Antibody dilutions were: anti-HIF-1α 1:350, anti-COX-2 1:100, anti-Met 1:200, anti-HGF 1:200, anti-CXCR4 5µg/ml; anti-VEGF 1:200. Densitometric analysis was performed after reaction with ECL-or ECL plus-chemiluminescence kit (GE Healthcare, Milan, Italy).

ELISA Assay

TGF-1 and HGF were measured in conditioned medium of cells exposed to hypoxia for 24 h in the absence of serum, following manufacturer's protocol.

Statistical Analysis

Luciferase activities and densitometric values were analyzed by analysis of variance, with P < 0.05 considered significant. Differences from controls were evaluated on original experimental data.

COX-2 half-life was evaluated using densitometric values of Western blot by the least squares method.

Results

Involvement of COX-2-signaling pathway in the hypoxic response

Bone is a hypoxic microenvironment capable of potentiating tumor metastatic growth [START_REF] Kingsley | Molecular biology of bone metastasis[END_REF].

Previous reports have shown that HIF-1α overexpression is correlated with distant metastasis and poor prognosis for breast cancer patients, and is more frequently observed in metastases than in primary tumor, suggesting an active role of HIF-1α in regulating metastatic progression [START_REF] Zhong | Overexpression of hypoxia-inducible factor 1α in common human cancers and their metastases[END_REF]. In addition, HIF-1α seems to be a promising target for controlling organotropic metastatization of breast cancer [START_REF] Lu | In vivo dynamics and distinct functions of hypoxia in primary tumor growth and organotropic metastasis of breast cancer[END_REF], and inhibitors of HIF-1α and TGF-β may improve treatment of bone metastases obtained with MDA-MB231 cells increasing patient survival [START_REF] Kang | A multigenic program mediating breast cancer metastasis to bone[END_REF][START_REF] Hiraga | Stimulation of cyclooxygenase-2 expression by bone-derived transforming growth factor-beta enhances bone metastases in breast cancer[END_REF][START_REF] Dunn | Hypoxia and TGF- drive breast cancer bone metastases through parallel signaling pathways in tumor cells and the bone microenvironment[END_REF]. TGF-β and HGF regulate COX-2 expression in breast and colon carcinoma cells, respectively, and hypoxia potentiates HGFdependent PGE 2 release in colorectal cancer [START_REF] Hiraga | Stimulation of cyclooxygenase-2 expression by bone-derived transforming growth factor-beta enhances bone metastases in breast cancer[END_REF]32]. PGE 2 participates in nuclear translocation of exogenously expressed HIF-1α in prostate carcinoma cells [START_REF] Liu | Prostaglandin E 2 induces hypoxia-inducible factor-1α stabilization and nuclear localization in human prostate cancer cell line[END_REF]. COX-2 inhibition largely prevents MDA-MB231 bone metastasis by decreasing osteoclast number and increasing apoptosis [START_REF] Hiraga | Stimulation of cyclooxygenase-2 expression by bone-derived transforming growth factor-beta enhances bone metastases in breast cancer[END_REF].

Here, we extended and deepened the knowledge on the molecular events of breast cancer metastatization by evaluating the involvement of COX-2-signaling pathway downstream of biological signals (TGF-β1 and HGF) in the hypoxic response, and we compared in vivo and in vitro models-using the 1833 bone metastatic clone-and human bone metastasis.

As shown in Fig. 1a, hypoxia increased COX-2 and HIF-1α fluorescence signals in nuclei of human 1833 cells, index of their co-localization (see merge images), and COX-2 signal also in perinuclear compartment. The treatment with NS398, a COX-2 inhibitor, largely reduced COX-2 expression throughout the cell, and prevented nuclear HIF-1α translocation. A more quantitative evaluation of HIF-1α protein was obtained by Western blot (Fig. 1b). HIF-1α-protein level increased (about 4-fold) in hypoxic-1833 cells, and NS398 almost completely blocked this stimulatory effect. Consistently, hypoxia treatment increased sixfold HRELuc activity in1833 cells that was counteracted by NS398 (Fig. 1c).

COX-2 and HIF-1α expression in 1833-xenograft model of metastasis

We evaluated metastases 20 days after intracardiac-cell injection (Fig. 2). COX-2 localized in perinuclear/cytosolic and nuclear compartments of bone-metastatic cells. HIF-1α localized prevalently in the cytosol, and also 20% of the nuclei of bone metastases were positive (60X magnification, inset). In addition, specific-HIF-1α signal was detected in bone-marrow microenvironmental cells (20X magnification), possibly as consequence of metastasis engraftment.

In fact, in control animals HIF-1α specific signal was undetectable. HIF-1α induction might depend on hypoxic conditions and/or growth-factor production [START_REF] Desiderio | Hepatocyte growth factor in invasive growth of carcinomas[END_REF][START_REF] Eliasson | The hematopoietic stem cell niche: low in oxygen but a nice place to be[END_REF].

Association of COX-2 and HIF-1α in human specimens of bone metastasis COX-2 and HIF-1α association has never been investigated in human bone metastasis from breast carcinoma. Fig. 3a shows the immunohistochemistry of COX-2 and HIF-1α in specimens from 2 patients, representative of 10 patients examined. Immunohistochemical images for patients 3 to 10 are given in Supplementary material. For all the patients examined, the percentage of cells positive for both COX-2 and HIF-1α was more than 70%. The presence of breast-carcinoma metastasis in the bones was evaluated by H&E staining, and by positive immunoreactivity for cytokeratines.

As shown in Fig. 3a andb, COX-2 accumulated predominantly in perinuclear and cytosolic compartments, but also a specific immunoreactivity in the nucleus was observed. HIF-1α in patients 1, 6, 8 and 9 completely localized in the nuclei; prevalent nucleic but also cytosolic HIF-1α immunostaining was observed in the other patients (Fig. 3a andb; Supplementary material). Thus, 100% of the examined patients showed nuclear localization of HIF-1α in bone metastases. The concomitant presence of COX-2 and HIF-1α in nuclei might indicate their functional interaction, consistent with the in vitro experiments showing COX-2 and HIF-1α co-localization as well as the regulation of HIF-1α translocation and HIF-1 activity by COX-2.

1833 clone and MDA-MB231-parental cells differently responded to hypoxia and TGF-1

We investigated the molecular mechanism(s) involved in the control of COX-2 expression by hypoxia, the TGF-1 cross-talk, and the possible differences between 1833 and MDA-MB231 cells.

As shown in Fig. 4a, in stimulated 1833 cells COX-2-protein levels increased between 12 and 24 h: the effect of hypoxia (13-14-fold increase) was more than that of TGF-1 (about 5-6-fold increase). Differently, in MDA-MB231 cells TGF-1 progressively enhanced COX-2-protein levels, reaching the maximum at 12 h (6.5-fold increase), while hypoxia was ineffective. The gels for the two cell lines were run in parallel and developed concomitantly, showing that the COX-2protein level was 4-fold higher in MDA-MB231 than in 1833 cells.

The half-life of COX-2 was measured in the two cell lines under the studied stimuli, blocking protein synthesis with cycloheximide (Fig. 4b). Cycloheximide (100 µg/ml) was added to the cells exposed to 12-h hypoxia or TGF-1, and Western blots were performed using total-cell extracts (not containing nuclear proteins). Densitometric values were analysed by the least-squares method. In 1833 cells, hypoxia markedly reduced the half-life of COX-2: it was 24 h in control cells diminishing to 2 h under hypoxia. TGF-1 practically unaffected the control value. In MDA-MB231 cells, TGF-1 reduced COX-2 half-life: it was 48 h in control cells and 13.5 h in TGF-1treated cells.

To better clarify the strong decrease of COX-2 half-life in hypoxic-1833 cells, we performed immunofluorescence experiments (Fig. 4c). Hypoxia increased COX-2-fluorescence signal in nuclear and perinuclear compartments, but after 2-h cycloheximide treatment the enhanced fluorescence signal localized almost completely in the nuclei. A partial redistribution both in nucleus and cytosol occurred in 4-h cycloheximide treated cells. Thus, the rapid decline of COX-2 protein after cycloheximide, in the study of half-life, might largely depend on nuclear translocation of COX-2 in 1833 cells.

Further studies were performed with LLnL, a specific proteasome inhibitor (Fig. 4d). LLnL stabilized COX-2 between 6 and 24 h in hypoxic 1833 cells and in TGF-1-treated MDA-MB231 cells, possibly by preventing proteasomal degradation.

TGF-1 and HGF controlled the response to hypoxia in bone metastasis of breast cancer We extended and deepened the studies on the role of growth factors in the hypoxic response by considering whether they were involved in COX-2 transcription (Fig. 5). COX-2 transactivating activity was examined using two gene-reporters, that contained sequences spanning from -1432 to +59 and from -327 to +59. The latter contains 4 Ets-1 and 1 NF-kB consensus sequences. COX-2(-1432/+59)Luc shows upstream 1 consensus site for each of the following transcription factors: TCF, HIF-1, AP-1 and NF-kB [START_REF] Lee | Hepatocyte growth factor regulates cyclooxygenase-2 expression via -catenin, Akt, and p42/p44 MAPK in human bronchial epithelial cells[END_REF].

As shown in Fig. 5a, in 1833 and MDA-MB231 cells TGF-1 increased luciferase activity of both the constructs, while hypoxia was ineffective. The stimulatory effect of the combined stimuli seemed, therefore, to be due to TGF-1. Basal activities of COX-2-promoter constructs (-1432/+59)Luc and (-327/+59)Luc were 2-and 1.5-fold higher in 1833 clone than in MDA-MB231 cells (data not shown).

HGF enhanced (about 2.5-fold) COX-2(-327/+59)Luc activity in 1833 but not in MDA-MB231 cells (Fig. 5b). However, HGF increased COX-2 protein levels between 6 and 24 h in the two cell lines, but with different time-courses and extent. The differences in the COX-2-protein levels might depend on the higher basal level in MDA-MB231 than in 1833 cells, and on the involvement of transcription-independent mechanism(s), such as protein stabilization by HGF in MDA-MB231 cells (data not shown).

The release of TGF-1 and HGF augmented of about 5-and 2-fold in the conditioned medium of 1833 cells exposed to hypoxia, while only TGF-1 release doubled in hypoxic MDA-MB231 cells (Fig. 5c).

Altogether, the data suggested that hypoxia did not act directly at transcriptional level, but COX-2 induction in 1833 cells was mediated by TGF-1 and HGF. Translational and posttranslational mechanisms seemed to participate in COX-2 protein accumulation in hypoxic-bone metastatic cells.

Then, we considered the transcription factors involved in COX-2 transactivation in response to the growth factors (Fig. 5d). First the cells, transfected with COX-2(-1432/+59)Luc, were exposed or not to TGF-1 in the presence of expression vectors for dominant negatives or specific inhibitors of the transcription factors showing consensus sequences in the promoter. In 1833 cells, ARNT, Ets1 or CGP049090 reduced basal and TGF-1-stimulated luciferase activity, while TAM67 prevented of about 60% luciferase activity after TGF-1 exposure. In MDA-MB231 cells, SN50, TAM67 or CGP049090 reduced basal and TGF-1-stimulated luciferase activity, while Ets1 prevented of about 95% luciferase activity after TGF-1 exposure. ARNT was ineffective in MDA-MB231 cells. In further experiments, in 1833 cells we examined COX-2(-327/+59)Luc activity, that was responsive to HGF. ΔEts1 reduced basal and HGF-stimulated luciferase activity, while SN50 showed an inhibitory effect only after HGF exposure.

Mechanisms of COX-2 induction by HGF

To further evaluate the mechanisms of COX-2 induction by HGF in 1833 cells, we examined whether the increase in COX-2 protein levels depended on stabilization beyond transcription. 1833 cells were exposed to HGF also in the presence of LLnL. As shown in Fig. 6a, HGF increased COX-2-fluorescence signals in perinuclear and nuclear compartments. HIF-1α showed a strong nuclear signal, and co-localized with COX-2 in this compartment. LLnL pre-treatment principally enhanced COX-2, showing strong fluorescence signals at perinuclear and nuclear levels. Western blot analysis was consistent with the stabilization of COX-2 protein, enhanced by HGF, after proteasome blockade (Fig. 6b). The half-life of COX-2 was unchanged by HGF treatment (Fig. 6c).

In HGF-treated 1833 cells (Fig. 6d), the activities of gene reporters driven by NF-kB and Ets1 multimers increased 2.2-fold and 4.2-fold, respectively, confirming their role in COX-2 transcription.

Effect of TGF-1 and hypoxia on transcription-factor activities

Based on the data obtained with dominant negatives and specific inhibitors, we examined the transcription factors in response to TGF-1 or hypoxia. HIF-1 is an α/ heterodimer [START_REF] Semenza | HIF-1 and mechanisms of hypoxia sensing[END_REF], and the regulation of the expression of these subunits might depend on the stimuli and the cell type. Under hypoxia, HIF-1α-protein level while augmenting (5-fold) in 1833 and MDA-231BO cells, only doubled in MDA-MB231 cells (Fig. 7a). The HIF-1β/ARNT-wild type form (97 kDa) was predominant in nuclei of the two bone-metastatic clones, increasing after hypoxia. In invasive MDA-MB231 cells HIF-1β/ARNT protein level, low in control nuclei, was reduced (-85%) by hypoxia (Fig. 7a). Low-molecular-weight (36 kDa) variant of HIF-1β/ARNT was expressed in parental MDA-MB231 cells and in the subclones (data not shown).

In 1833 cells, hypoxia stimulated HIF-1 activity (6-fold increase in HRELuc) (Fig. 7b HRELuc was activated in hypoxic MDA-231BO cells, being practically unmodified by HIF-1β-expression vector transfection (Fig. 7d). HIF-1α controlled transcription factor activities TGF-1 and HGF enhanced HIF-1α protein level in 1833 cells, while decreasing that of MDA-MB231 cells (Fig. 8a andb). We reproduced endogenous-HIF-1α overexpression by transfecting HIF-1α-expression vector, and we studied the activities of the gene reporters driven by multimers of the consensus sequences. The transcription factors examined were activated by HIF-1α overexpression, except TCF. In fact, TOPFLASHLuc was reduced of about 60% (Fig. 8c). It is worth noting that the activity of the HRE multimer, very elevated under basal conditions, doubled in 1833 cells transfected with HIF-1α. This overexpression of the exogenous α subunit seemed to partly reproduce the effect of TGF-1 in the regulation of COX-2 transcription, because HIF-1 activation is complex requiring translocation and phosphorylation of the α subunit, beyond stabilization, and the possible co-operation with other transcription factors and transcriptional coactivators [35].

Target genes controlled by COX-2-signaling pathway, and possible interaction with HGF/Met system The gene expression pattern of 1833 cells, under TGF-1 or hypoxia exposure, was examined after treatment with NS398 or SB431542 to evaluate the involvement of COX-2 signaling and TGF-1-typeI receptor, respectively. In Fig. 9a, the results of experiments performed with total protein extracts are reported. TGF-1 and hypoxia increased HGF protein levels. The HGF-protein expression was enhanced by NS398 or SB431542 in the presence of TGF-1. In contrast, TGF-1-typeI receptor blockades reduced HGF-protein level under hypoxia. Met 170 and 145 kDa were the precursor and the membrane-spanning subunit, respectively. Hypoxia-stimulatory effects on Met and VEGF were partly prevented by COX-2 and TGF-1-typeI receptor blockades, respectively.

For nuclear extracts (Fig. 9b), differences were observed between control (10% FBS) and starved cells depending on the gene. CXCR4-protein level was reduced (from 50 to 80%) by hypoxia, with reversion after TGF-1-typeI receptor blockade, and was unchanged by TGF-1 treatment, in the presence or the absence of the inhibitors. Noteworthy, CXCR4-protein level was very faint using total extracts (data not shown). Nuclear COX-2 was induced by hypoxia, and COX-2 and TGF-1-typeI receptor blockades oppositely affected COX-2 levels. In particular, NS398 enhanced nuclear COX-2 protein level between 6 and 18 h of hypoxia, while decreasing hypoxic effect (-45%) at 24 h. The latter data was in agreement with immunofluorescence experiments (Fig. 1a). HIF-1α induction by both stimuli was prevented by the inhibitors used.

Summary of molecular events involved in COX-2/HIF-1α interaction in bone metastasis

In Fig. 10 we show TGF-1 and HGF autoregulatory loops, triggered in response to hypoxia, that were responsible for COX-2 expression and HIF-1 activity, controlling a panel of genes relevant for the bone-metastatic process.

Discussion

Physical (hypoxia) and biological (growth factors) stimuli of bone-metastasis microenvironment seemed to favour COX-2-HIF-1 functional interaction, thus affecting gene expression in breast carcinoma metastatic cells and human metastasis with bone avidity. This was suggested by data obtained with COX-2 inhibitor, blocking HIF-1 transactivating activity, and by the observed nuclear COX-2-HIF-1α co-localization, using different in vitro and in vivo models and human specimens.

First, our study extended the knowledge of the molecular events involved in bone metastatisation, and demonstrated that 1833-bone metastatic clone differed from highly-invasive parental MDA-MB231 cells not only for gene-expression level [START_REF] Kang | A multigenic program mediating breast cancer metastasis to bone[END_REF], but especially for the remarkable plasticity and adaptability of the gene profile to multiple-concomitant microenvironmental stimuli. This depended principally on functionality of HIF-1-transcription factor in 1833 cells, in agreement with our previous work [START_REF] Bendinelli | NF-kB activation, dependent on acetylation/deacetylation, contributes to HIF-1 activity and migration of bone metastatic breast carcinoma cells[END_REF], a molecular characteristic shared with MDA-231BO cells also endowed of bone tropism.

Metastatic cells might produce HGF and TGF-1 in bone-hypoxic environment, and HGF might substitute for TGF-1 during metastasis progression. In fact, TGF-1 seemed to exert a negative effect on HGF signaling, because TGF-1-typeI receptor blockade enhanced HGF-protein level in 1833 cells. Thus, the metastasis-gene signature was likely to be strongly controlled at the secondary-bone site, undergoing changes in stimuli composition after metastatic-cell engraftment [START_REF] Previdi | Interaction between human-breast cancer metastasis and bone microenvironment through activated hepatocyte growth factor/Met and -catenin/Wnt pathways[END_REF]. The composition of bone microenvironment may vary depending on the growth phase from early metastasis, leaving dormancy, until acquisition of a more aggressive phenotype during metastasis progression. Consistently, elevated HIF-1α expression was observed in cells of metastasis microenvironment of the xenograft model, also supporting the statement: metastatic cells in the bone exploit surrounding cells to their own ends, resulting in invasion, neoangiogenesis and ultimately in inappropriate growth and metastasis establishment [START_REF] Joyce | Microenvironmental regulation of metastasis[END_REF]. Therefore, in our model HIF-1α expression was not limited to 1833 metastasis, but participated to supportive-cell function, compatible with a "pre-metastatic niche". Altogether, these properties probably render so difficult to therapeutically intervene halting bone metastases.

Second, we identified the molecular mechanisms of hypoxia as leading stimulus in bone metastasis. Through HGF and TGF-1 release, possibly activating autoregulatory loops with Met and TGF-1 receptors, hypoxia affected COX-2 and the gene-pattern downstream including HIF-1α. COX-2 and HIF-1α genes were co-expressed in nuclei of human-bone metastasis specimens, index of reciprocal influence. Studies in vitro showed, in fact, that COX-2 participated in HIF-1αnuclear translocation in hypoxic-1833 cells, and the shuttling of endogenously overexpressed HIF-1α was critical for HIF-1 activity. COX-2 blockade prevented both these molecular events.

The precise molecular roles played by COX-2 and HIF-1 in the metastatic process are here clarified. COX-2 expression and localization were deeply studied in a wide number of specimens of human breast-carcinoma bone metastasis. Previously, Hiraga et al. [START_REF] Hiraga | Stimulation of cyclooxygenase-2 expression by bone-derived transforming growth factor-beta enhances bone metastases in breast cancer[END_REF] evaluated COX-2 by immunohistochemistry in 1 bone metastasis from breast cancer, suggesting an involvement of COX-2 in osteolysis using an animal model. Because the cytoplasm is the site of new-protein synthesis, COX-2 accumulation in the nucleus of human-bone metastasis was indicative of active COX-2-nuclear import in transcriptionally activated cells. Perichromatin regions of the nucleus are sites of active transcription [START_REF] Parfenova | Dynamics of nuclear localization sites for COX-2 in vascular endothelial cells[END_REF]. HIF-1 functioned in hypoxic 1833 and MDA-231BO but not in MDA-MB231 cells. The MDA-MB231 cells have a mutated HIF-1β/ARNT subunit [START_REF] Wilson | Aryl hydrocarbon (Ah) nonresponsiveness in estrogen receptor-negative MDA-MB231 cells is associated with expression of variant Arnt protein[END_REF], and a reduced level of the wild-type form was observed by us under hypoxia. The data were consistent with an alternative-RNA splicing in hypoxic conditions reducing the level of HIF-1β/ARNT wildtype protein [START_REF] Hirschfeld | Alternative splicing of Cyr61 is regulated by hypoxia and significantly changed in breast cancer[END_REF].

A strong HIF-1α-nuclear signal was observed in all the human-metastasis specimens. HIF-1α and COX-2 co-localization in nuclei of human-bone metastasis not only validated in vivo the functional interaction demonstrated in vitro, but might have also a diagnostic significance for metastasis relapse after therapy, and prognostic significance to individuate primary tumor. HIF-1α localizes in nuclei of bone metastasis of prostate carcinoma [START_REF] Zhong | Overexpression of hypoxia-inducible factor 1α in common human cancers and their metastases[END_REF], but a concomitant COX-2-nuclear expression and functional interaction are not shown.

In primary lobular and ductal mammary tumors, COX-2 is present in plasma-membrane caveolae and cytosol, respectively [START_REF] Perrone | COX-2 localization within plasma membrane caveolae-like structures in human lobular intraepithelial neoplasia of the breast[END_REF], without the nuclear and perinuclear localization observed by us in bone metastasis.

The regulation of COX-2 in 1833 cells was complex. Hypoxia regulated indirectly COX-2 transactivation via HGF and TGF-1, controlling the activity of specific transcription factors that bind to COX-2 promoter, i.e. AP-1 and NF-kB, while HIF-1, Ets1 and β-catenin/TCF controlled both basal and activated COX-2 transactivation. HIF-1α protein, stabilized by HGF and TGF-1, regulated the network of transcription factors including HIF-1. Thus, HIF-1 activity was important for COX-2 transactivation because the blockade of HIF-1β/ARNT with the dominant negative largely prevented luciferase activity. As other early genes, COX-2 is regulated not only at transcriptional level but also at translational and post-translational levels [START_REF] Singh-Ranger | The role of cyclooxygenase-2 in breast cancer: review[END_REF]. A rapid downregulation of cellular COX-2 occurring under hypoxia in 1833 cells, depended on nuclear translocation and proteasomal degradation, mechanisms important to control COX-2-signaling function on gene expression.

Altogether, the relevance of our work was that COX-2 signaling regulated HIF-1 activity by controlling HIF-1α expression and nuclear translocation. Active-transcription factors are localized in nuclear matrix, where signal-transduction mechanism(s) are working [START_REF] Parfenova | Dynamics of nuclear localization sites for COX-2 in vascular endothelial cells[END_REF]. The blockade of enhanced TGF-1-typeI receptor prevented COX-2 and HIF-1α expression in nuclei, consistent with an enhancement of TGF-1/TGF-1-typeI receptor couple, condition necessary for the autoregulatory-loop triggering [START_REF] Li | Effects of hypoxia on expression of transforming growth factor-1 and its receptors I and II in the amoeboid microglial cells and BV-2 cells[END_REF].

Based on this complex orchestration of signaling pathways, 1833-bone metastasis possibly differed from MDA-MB231-bone metastasis, in which TGF-1 seems the only stimulus responsible for COX-2 expression and function [START_REF] Hiraga | Stimulation of cyclooxygenase-2 expression by bone-derived transforming growth factor-beta enhances bone metastases in breast cancer[END_REF]. In cells practically unresponsive to hypoxia such as MDA-MB231, being HIF-1 unaffected, TGF-1 principally induced NF-kB. The ligand TGF-1 signals from the receptor to the nucleus using a set of proteins termed Smads, and NF-kB might exert inhibitory roles blocking Smad phosphorylation [START_REF] Roman-Blas | Modulation of TGF- signaling by proinflammatory cytokines in articular chondrocytes[END_REF]. Consistently, we observed that HIF-1α, a target gene of NF-kB [START_REF] Tacchini | Hepatocyte growth factor signalling stimulates hypoxia inducible factor-1 (HIF-1) activity in HepG2 hepatoma cells[END_REF], was down-regulated in TGF-1-exposed MDA-MB231 cells. Our present data possibly explain why knockdown of HIF-1α has not additive effects with TGF- blockade against MDA-MB231-bone metastasis [START_REF] Dunn | Hypoxia and TGF- drive breast cancer bone metastases through parallel signaling pathways in tumor cells and the bone microenvironment[END_REF]. In the case of 2-metoxyestradiol (2ME2), showing a relatively small-inhibitory effect on hypoxia-induced HIF-1-transcriptional activity [START_REF] Chua | Structure activity analysis of 2-methoxyestradiol analogues reveals targeting of microtubules as the major mechanism of antiproliferative and proapoptotic activity[END_REF], it affects bone-metastasis burden possibly as anticancer agent with antiproliferative, antiangiogenic and proapoptotic roles by disrupting the microtubule skeleton. Only after exogenous Smads overexpression, VEGF transactivation increases in response to TGF- [START_REF] Dunn | Hypoxia and TGF- drive breast cancer bone metastases through parallel signaling pathways in tumor cells and the bone microenvironment[END_REF].

Third, due to the relationship with HIF-1α/HIF-1 activity and considering the HIF-1-target genes, completely new functions beyond osteolysis can be proposed for COX-2 in human-bone metastasis, such as angiogenesis (VEGF), homing (CXCR4) and protection from apoptosis. HIF-1α is an anti-apoptotic gene [START_REF] Semenza | HIF-1 and mechanisms of hypoxia sensing[END_REF][START_REF] Rankin | The role of hypoxia-inducible factors in tumorigenesis[END_REF][START_REF] Marignol | Hypoxia in prostate cancer: a powerful shield against tumour destruction?[END_REF], while COX-2 is pro-apoptotic [START_REF] Lin | The pro-apoptotic action of stilbene-induced COX-2 in cancer cell: convergence with the antiapoptotic effect of thyroid homone[END_REF]. Opposite regulation of HIF-1α-and COX-2-protein levels was observed in nuclei of hypoxic-1833 cells under COX-2signaling blockade, as a possible consequence of a feed-back control mechanism that enhanced COX-2-protein level in the absence of HIF-1α protein.

Hypoxia decreased nuclear CXCR4-protein level in 1833 cells through TGF-1-typeI receptor and COX-2. CXCR4 down-regulation might be important for the homing of metastatic cells, preventing their recirculation, while CXCL12 drives them to bone-secondary site. CXCR4 in breast cancer tissues shows a variable subcellular localization, e.g. on the plasma membrane, in the cytoplasm, or even in the nucleus [START_REF] Schmid | CXCR4 is expressed in ductal carcinoma in situ of the breast and in atypical ductal hyperplasia[END_REF]. CXCR4-nuclear localization occurs also in hemangioblastoma and clear cell-renal carcinoma [START_REF] Zagzag | Stromal cell-derived factor-1α and CXCR4 expression in hemangioblastoma and clear cellrenal cell carcinoma: von Hippel-Lindau loss-of-function induces expression of a ligand and its receptor[END_REF]. Various receptors are known to function intranuclearly, possibly affecting specific signaling pathways [START_REF] Matteucci | Nuclear localization of active HGF receptor Met in aggressive MDA-MB231 breast carcinoma cells[END_REF].

The VEGF expression in metastatic cells, under the influence of microenvironment stimuli that activated HIF-1, might be important for neoangiogenesis at the secondary-growth site. The blockade of TGF-β1-typeI receptor reduced VEGF expression in 1833 cells. Surprisingly, in TGF-1-treated 1833 cells a negative interrelation occurred between TGF-1 and HGF expression, as previously reported for fibroblast-HGF and tumor cell-TGF-1 [START_REF] Matsumoto | Hepatocyte growth factor and the Met system as a mediator of tumor-stromal interactions[END_REF], and COX-2 influenced HGF-protein level. Vice versa, HGF regulated COX-2 via Ets-1 and NF-kB.

The key role exerted by microenviromental stimuli on metastatic phenotype may be of great importance from a therapeutic point of view, because it would be more useful and easier to affect gene profile at epigenetic level than to modify the genotype. Even if hypoxia-response gene signature is not able to stratify patients for risk of bone metastasis, based on gene expression profile of primary-breast tumor, the strategy of targeting HIF-1α for treatment of bone metastasis remains valid [START_REF] Lu | In vivo dynamics and distinct functions of hypoxia in primary tumor growth and organotropic metastasis of breast cancer[END_REF]. We demonstrated that hypoxia led to an adaptive response via COX-2, orchestrated by HIF-1, that might be crucial for metastasis progression and therapy resistance, responsible for poor patient outcome. Downstream targets of HIF-1α/HIF-1 would be considered as hypoxia biomarkers, and the identification of the molecular biomarkers COX-2 and HIF-1α, with a potential to predict metastasis-treatment outcome, is essential for selecting patients to receive the most beneficial therapy.

  ), while decreasing TCF activity (-40% TOPFLASHLuc) (Fig 7c). In TGF-1-treated 1833 cells, NF-kB, AP-1 and Ets-1 activities increased (2-3-fold) (Fig 7c). In MDA-MB231 cells, HIF-1βexpression vector transfection rescued HRELuc activity under hypoxia (Fig 7b), and TGF-1 tripled NFkBLuc and doubled TOPFLASHLuc (Fig 7c).

Fig. 4

 4 Fig. 4 Regulation of COX-2-protein levels by microenvironmental stimuli. a Time-courses of

Fig. 5

 5 Fig.5COX-2 transactivation after TGF-β1, HGF and hypoxia. In a and b, the cells were

Fig. 8

 8 Fig. 8 Cellular HIF-1α-expression under microenvironmental stimuli, and role in the control of

Fig. 9

 9 Fig. 9 Genes regulated by COX-2 and TGF-β1-typeI receptor. The numbers at the bottom of the

Fig. 10

 10 Fig. 10 Scheme representing the role of TGF-β1 and HGF released by 1833 cells under hypoxia

Acknowledgments

This study was supported by founds from Ministero Istruzione-Università-Ricerca, PRIN-2007-12-25201001-14; Ministero Salute, Ricerca Corrente-4029.

Legends to Figures