Rabab A A Mohammed Ae

Ian O Ellis

A E Somaia

Elsheikh Ae

Emma C Paish

A E Stewart
email: stewart.martin@nottingham.ac.uk

G Martin

R A A Mohammed

Á S G Martin

Á S Elsheikh

Á E C Paish

Lymphatic and angiogenic characteristics in breast cancer: morphometric analysis and prognostic implications

Keywords: Angiogenesis, Breast cancer, D2-40, Lymphangiogenesis and prognosis

Controversy exists regarding the topography of lymph vessels in breast cancer, their usefulness as prognostic factors, relationship with angiogenesis and whether active lymphangiogenesis occurs within the tumour. A series of 177 well-characterized breast cancers, with long term follow up, were stained with D2-40, CD31 and CD34. Distribution of lymphatics and lymph vessel density (LVD) were assessed in three areas, intratumoural, peripheral and peritumoural and correlated with clinicopathological criteria and patient prognosis. Microvessel density (MVD) was assessed and correlated with LVD. Double immunohistochemical staining with D2-40 and MIB-1 was carried out to assess the proliferative status of lymphatics and of the tumour emboli within. Peritumoural lymphatics were detected in all tumours whereas peripheral and intratumoural lymphatics were detected in 86 and 41% of specimens, respectively. Tumours with higher total LVD were significantly associated with the presence of lymph node (LN) metastasis and shorter overall survival (OS). In multivariate analysis, tumour grade, LN status and the presence of lymphovascular invasion, but not LVD, were independent poor prognostic factors. No association was found between LVD and MVD. Proliferating lymphatics were detected in 29% of specimens and were significantly associated with dense inflammatory infiltrate. In conclusion, lymphatics are located primarily in the peritumoural and peripheral areas in breast cancer and seem to play an important role in disease progression by being routes for tumour dissemination. The lack of correlation between lymphangiogenic and angiogenic characteristics suggests two distinct processes and the presence of active lymphangiogenesis, albeit in a small portion of specimens, may have important therapeutic implications.

Introduction and aims

Although the lymphatic system was first described by the Italian anatomist Asellius in 1627, as vein-like vessels carrying whitish fluid, research in this area has progressed slowly compared with that in blood vessels [START_REF] Hong | Development of the lymphatic vascular system: a mystery unravels[END_REF]. One of the major limitations has been the lack of histological, ultrastructural, and immunohistochemical markers that can accurately differentiate blood endothelial cells (BECs) from lymphatic endothelial cells (LECs). Recently molecular lymphatic markers have been characterized and used successfully to differentiate lymphatics from blood vessels. These include podoplanin/D2-40, LYVE-1, Prox-1, mannose receptor and VEGFR-3. Several articles and reviews have been published describing their structures, functions and roles in lymphangiogenesis [START_REF] Ma | Molecular and cellular mechanisms of lymphangiogenesis[END_REF][START_REF] Banerji | LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan[END_REF][START_REF] Breiteneder-Geleff | Podoplanin, novel 43-kd membrane protein of glomerular epithelial cells, is down-regulated in puromycin nephrosis[END_REF][START_REF] Chilosi | New diagnostic markers: podoplanin-d2-40[END_REF][START_REF] Ferrara | The biology of vascular endothelial growth factor[END_REF][START_REF] Hong | Prox1, master regulator of the lymphatic vasculature phenotype[END_REF].

Characterization of these molecules has lead to a surge in research investigating the role of lymphatics in normal physiology and in diseases, particularly in cancer progression. Clinicopathological data suggest that lymphatics are most likely to be the initial route for the spread of certain solid tumours, such as breast cancer [START_REF] Mohammed | Improved methods of detection of lymphovascular invasion demonstrate that it is the predominant method of vascular invasion in breast cancer and has important clinical consequences[END_REF]. Although numerous studies have investigated lymphatics in breast cancers, many questions remain unanswered. There is still debate whether lymphatics are present within tumours or located only at the periphery. Equally, debate still occurs on whether active lymphangiogenesis is a feature in breast cancer.

Initial studies on xenografted tumours reported that lymphatics were only present in peritumoural areas and absent from the intratumoural compartment [START_REF] Leu | Absence of functional lymphatics within a murine sarcoma: a molecular and functional evaluation[END_REF][START_REF] Padera | Lymphatic metastasis in the absence of functional intratumor lymphatics[END_REF]. Such studies also suggested that these vessels were the main route for metastasis and that lymphangiogenesis did not seem to play a role in this process. Opposing results have been reported by later studies where functional intratumoural lymphatics were detected in xenografted tumours [START_REF] Pathak | Visualizing function in the tumor-associated lymphatic system[END_REF]. With human breast cancer certain studies have reported an absence of both intratumoural lymphatics and of lymphangiogenesis [START_REF] Agarwal | Lymphangiogenesis does not occur in breast cancer[END_REF][START_REF] Vleugel | Lack of lymphangiogenesis during breast carcinogenesis[END_REF][START_REF] Williams | Absence of lymphangiogenesis and intratumoural lymph vessels in human metastatic breast cancer[END_REF] whereas others have detected lymphatics both inside and around the tumour [START_REF] Van Der Auwera | Tumor lymphangiogenesis in inflammatory breast carcinoma: a histomorphometric study[END_REF][START_REF] Choi | Angiogenic and lymphangiogenic microvessel density in breast carcinoma: correlation with clinicopathologic parameters and VEGF-family gene expression[END_REF][START_REF] Bono | High LYVE-1-positive lymphatic vessel numbers are associated with poor outcome in breast cancer[END_REF]. The importance of the lymph vessel density (LVD) in promoting metastatic spread of breast cancer is questionable [START_REF] Kato | A quantitative analysis of lymphatic vessels in human breast cancer, based on LYVE-1 immunoreactivity[END_REF][START_REF] Nakamura | Lymph vessel density correlates with nodal status, VEGF-C expression, and prognosis in breast cancer[END_REF] and which vessels, newly formed lymphatics or pre-existing ones, play the more important role in the initial dissemination of tumour cells is uncertain.

Detection of actively dividing LECs along lymph vessels has been considered a sign of active lymphangiogenesis. Double immunohistochemical staining of tumour sections with a lymphatic marker such as D2-40 or LYVE-1 and a proliferative marker such as Ki-67/MIB-1, or proliferating cell nuclear antigen (PCNA), have been used to address this question however, as with the aforementioned, results are controversial [START_REF] Van Der Auwera | Increased angiogenesis and lymphangiogenesis in inflammatory versus noninflammatory breast cancer by real-time reverse transcriptase-PCR gene expression quantification[END_REF][START_REF] Koukourakis | LYVE-1 immunohistochemical assessment of lymphangiogenesis in endometrial and lung cancer[END_REF].

We, like others, have shown in previous work that lymphangiogenesis and angiogenesis may share some common regulatory features [START_REF] Mohammed | Prognostic significance of vascular endothelial cell growth factors -A, -C and -D in breast cancer and their relationship with angio-and lymphangiogenesis[END_REF]. It is now appreciated that angiogenesis has an important role in the progression of breast cancer, through providing essential nutrients for tumour cell growth and proliferation [START_REF] Weidner | Tumor angiogenesis and metastasiscorrelation in invasive breast carcinoma[END_REF][START_REF] Bevilacqua | Prognostic value of intratumoral microvessel density, a measure of tumor angiogenesis, in nodenegative breast carcinoma-results of a multiparametric study[END_REF][START_REF] Toi | Tumour angiogenesis is an independent prognostic indicator in primary breast carcinoma[END_REF]. Microvessel density (MVD); as a measurement for angiogenesis, has been found to be strongly associated with features of tumour aggression as larger size and poor differentiation, however its role as a prognostic factor has not been firmly established [START_REF] Uzzan | Microvessel density as a prognostic factor in women with breast cancer: a systematic review of the literature and meta-analysis[END_REF].

The aims of the current study were (a) to study, in detail, the topography and characteristics of lymphatic vessels in breast cancer and examine their association(s) with clinicopathological criteria and influence on patient prognosis in a well characterized series of breast cancer with longterm follow-up, (b) to investigate the angiogenic characteristics in the same tumour specimens to discover if tumours share the common, and related, angiogenic and lymphangiogenic characteristics, and (c) to assess the proliferative status of tumoural lymphatics (as an indicator of lymphangiogenesis) and of tumour cells within lymphatics.

Material and methods

Patients and specimens

This study was conducted on pre-treatment primary invasive breast cancer specimens that have previously been described, in detail [START_REF] Mohammed | Improved methods of detection of lymphovascular invasion demonstrate that it is the predominant method of vascular invasion in breast cancer and has important clinical consequences[END_REF][START_REF] Mohammed | Prognostic significance of vascular endothelial cell growth factors -A, -C and -D in breast cancer and their relationship with angio-and lymphangiogenesis[END_REF]. Briefly, 177 consecutive paraffin-embedded archival specimens were obtained from the Department of Histopathology, Nottingham University Hospitals, City Hospital Campus. The median age of patients at time of diagnosis was 57 years (range, 32-70 years), 52 (29%) patients had positive lymph nodes with most tumours presenting as either stage I (n = 121 (68.4%)) or stage II (n = 43 (24.3%)) disease.

The clinical management of patients was based on tumour characteristics; by the Nottingham Prognostic Index (NPI) and by hormone receptor status. After surgical treatment, patients with an NPI score B3.4 received no adjuvant therapy, those with a NPI score [3.4 received Tamoxifen if oestrogen receptor (ER) positive (± Zoladex if pre-menopausal) or classical cyclophosphamide, methotrexate and 5-fluorouracil (CMF) if ER negative and fit enough to tolerate chemotherapy [START_REF] Madjd | High expression of Lewis y/b antigens is associated with decreased survival in lymph node negative breast carcinomas[END_REF]. Patients that received breast conserving surgery or mastectomy may receive a course of radiotherapy according to the risk of regional recurrence that is determined by the multidisciplinary team. Tumours were graded according to a modified Bloom-Richardson scoring system [START_REF] Elston | Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up[END_REF] and size was categorized according to the TNM staging criteria [START_REF] Singletary | Breast cancer staging: working with the sixth edition of the AJCC cancer staging manual[END_REF]. NPI was calculated as previously described [START_REF] Galea | The Nottingham Prognostic Index in primary breast cancer[END_REF]. Survival data, including survival time and disease free interval, was maintained on a prospective basis. The disease-free interval was defined as the interval (in months) from the date of the primary surgical treatment to the first loco-regional or distant recurrence. Overall survival was taken as the time (in months) from the date of the primary surgical treatment to the time of death. Complete clinical follow-up information was available for all 177 patients. The median follow-up period was 96 months (range, 2-184 months). Forty-one patients developed regional recurrence by the time of the last follow-up and sixteen patients died from the disease. Ethical approval for this study was obtained for analysis from Nottingham Local Research Ethics Committee (REC C2020313).

One hundred and thirty two of the patients (72.4%) were treated by surgery and subsequent adjuvant therapy. Of the 49 patients (27.6%) that did not receive any adjuvant therapy one died from the disease and eight developed a recurrence. Survival analysis was conducted only on those patients that received identical treatment i.e. those who were treated by surgery and adjuvant therapy. REMARK criteria were followed throughout, as recommended by McShane et al. [START_REF] Mcshane | REporting recommendations for tumor MARKer prognostic studies (REMARK)[END_REF].

Immunohistochemistry

For visualization of lymphatics and of blood vessels, 3 consecutive sections from each specimen were stained with the lymphatic marker D2-40 (SIGNET, 730-16, Cambridge, UK) and with blood vascular markers CD34 (SEROTEC, MCAP547, Oxford, UK) and CD31 (Dako-Cytomation, 0823, Denmark) as descried previously [START_REF] Mohammed | Prognostic significance of vascular endothelial cell growth factors -A, -C and -D in breast cancer and their relationship with angio-and lymphangiogenesis[END_REF].

For examination of the proliferative status of lymphatics, and of tumour cells within lymphatics, a double IHC technique with MIB-1 (Monoclonal Mouse Ki-67 Antigen, Clone MIB-1, Code-No. M 7240, DAKO, Denmark) and D2-40 was used. Briefly, 4 um-thickness sections were deparaffinized in xylene for 20 min and then were rehydrated by incubation in a sequence of descending concentration of Ethanol (*100, 70 and 50%). Sections were immersed in distilled water for 2 min. For antigen retrieval, sections were put in 0.01 mol/l sodium citrate buffer (pH 6.0) in microwave for 20 min before incubation in endogenous enzyme block solution, containing 0.5% hydrogen peroxide for 5 min to block any endogenous peroxidise activity. MIB-1 was then applied to sections for 1 h at room temperature (RT). Excess unbound antibody was washed with Tris-buffered saline (TBS). Sections were incubated in Polymer/HRP (a Dextran polymer conjugated with horseradish peroxidise and immunoglobulins) for 30 min at RT and then washed for 5 min with TBS. For visualization of MIB-1, sections were incubated in 5% 3,3 0diaminobenzidine tetrahydrochloride chromogen solution and substrate for 10 min and then washed with TBS for 5 min. Sections were then incubated for 3 min in blocking solution and then washed with TBS. Following blocking, D2-40 (monoclonal mouse AB) diluted in 1:100 was applied for 1 h at RT. In the next step Rabbit/Mouse (LINK) was added for 30 min and then sections were washed in TBS, followed by incubation with the Polymer/ AP reagent. To visualize the second staining with D2-40, Permanent Red Chromogen was applied for 5 min and then washed in running water for 3 min before counterstaining with haematoxylin and mounting with glycerol.

Assessment of lymphangiogenic and angiogenic characteristics

Two characteristics were noted for lymph vessels (1) distribution/location within and around the tumour and (2) total LVD. The majority of studies have assessed LVD by counting lymphatics in three or five hot spots (areas with highest density of lymphatics) [START_REF] Zeng | Lymphatic vessel density and lymph node metastasis in prostate cancer[END_REF][START_REF] Koskinen | Lymphatic vessel density in vocal cord carcinomas assessed with LYVE-1 receptor expression[END_REF][START_REF] Schoppmann | Lymphatic microvessel density and lymphovascular invasion assessed by anti-podoplanin immunostaining in human breast cancer[END_REF], in a fashion akin to that for the determination of MVD of vascular endothelium. As we were interested to examine density and distribution of lymphatics in the whole tumour, we used a modified method by counting all vessels in the whole section. First, each section was examined at low magnification (940) and divided into three areas, the central 2/3 core, the intratumoural (IT) area, the outer 1/3 of the tumour tissue, the peripheral tumoural area (PP) and the normal area surrounding the tumour, the peritumoural area (PT) as shown in Fig. 1. Vessels were counted in each area using 9100 magnification with a surface area of 3.46 mm 2 . The sum of LVD in all fields in each zone was divided by the total surface areas of all fields to give the LVD for that zone, presented as vessels/mm 2 . This method gave the LVD in the peritumoural area (LVD-PT), in the peripheral tumour area (LVD-PP), and in the intratumoural area (LVD-IT) with the sum of three giving the total LVD for the specimen. Although this method of assessment is difficult and time consuming it allows all lymphatics across the whole tumour section to be assessed thereby enabling distribution information to be obtained. Assessment of MVD was conducted using the Chalkely method as described previously [START_REF] Mohammed | Prognostic significance of vascular endothelial cell growth factors -A, -C and -D in breast cancer and their relationship with angio-and lymphangiogenesis[END_REF]. Lymphvascular invasion (LVI) was identified when tumour clusters were detected within D2-40-positive vessels.

Assessment of the proliferation fraction of lymphatics, of tumour emboli and of the invasive component of the tumour

Lymphatic nuclei were only scored as proliferating (MIB-1 positive) if they fulfilled features of an endothelial cell nucleus i.e. plump oval nuclei lying within the contour of the red LEC, Fig. 2f. Cells that did not fulfil such criteria, as shown in Fig. 2i, were excluded as they could represent Fig. 1 Tumour section with dashed lines showing how the section was divided into three areas; intratumoural (the inner 2/3 core of the tumour), peripheral (the outer 1/3 of the tumour) and peritumoural (all the normal tissue surrounding the tumour), magnification 920 a proliferating tumour or inflammatory cell infiltrating through the lymphatic vessel wall.

No standard method is currently used for the assessment of the proliferation fraction of lymphatics (PF-L) in human tumours. Different approaches have been used with certain studies simply reporting the presence or absence of MIB-1 positive LEC nuclei [START_REF] Beasley | Intratumoral lymphangiogenesis and lymph node metastasis in head and neck cancer[END_REF], with others reporting the number of positive nuclei/100 LECs nuclei [START_REF] Van Der Auwera | Increased angiogenesis and lymphangiogenesis in inflammatory versus noninflammatory breast cancer by real-time reverse transcriptase-PCR gene expression quantification[END_REF], and yet others counting the number of positive nuclei in three high power fields [START_REF] Koukourakis | LYVE-1 immunohistochemical assessment of lymphangiogenesis in endometrial and lung cancer[END_REF]. As both the proliferation fraction of the tumoural lymphatics (PF-L) and of the tumour emboli (PF-E) were to be assessed, specimens which had LVI were selected for the double IHC procedure; they were 54 specimens of which 41 specimens were characterized with high LVD. In seven of the 54 specimens, tumour emboli were small and lost by deeper sectioning of the paraffin blocks so that PF-E was assessed in 47 specimens. To allow statistical comparison between PF-L in specimens with high LVD and specimens with low LVD, an additional 31 specimens with low LVD were randomly selected from the study population giving a total of 85 specimens. PF-L was assessed by counting the total number of MIB-1 positive LEC nuclei in the whole tumour section and dividing it by the total LVD. A similar approach has been used previously [START_REF] Soares | Lymphatic vascular density and lymphangiogenesis during tumor progression of carcinoma expleomorphic adenoma[END_REF].

To assess the proliferation fraction of tumour emboli (PF-E) within the lymph vessels, the number of MIB-1positive tumour cell nuclei was divided by the number of total cells within the emboli. The proliferation fraction of the invasive component (i.e. not including ductal carcinoma in situ) of the tumour cells outside lymph vessels (PF-T), was scored using a method similar to that described by Potemski et al. and by Rudolph et al. [START_REF] Rudolph | Prognostic significance of Ki-67 and topoisomerase IIalpha expression in infiltrating ductal carcinoma of the breast. A multivariate analysis of 863 cases[END_REF][START_REF] Potemski | Ki-67 expression in operable breast cancer: a comparative study of immunostaining and a real-time RT-PCR assay[END_REF]. First the tumour fields with highest reactivity for MIB-1 were selected and then the number of MIB-1 positive tumour nuclei was counted in 500 tumour cells and this gave the percent of the proliferating tumour cells (500 tumour cells were present in 2-3 high magnification fields, 9400 according to amount of tumoural stroma). About 10% was chosen as a cut-off point to categorize the specimens into tumours with low PF-T and tumours with high PF-T.

A second observer re-examined 20% of specimens with good concordance between both readings; j value [85%.

Statistical analysis

For assessment of associations between LVD, MVD and clinicopathological criteria, specimens were categorized into two groups according to the median and then Chi squared and 2 9 2 tables were used to assess statistical significance. To assess prognostic significance of LVD and MVD, univariate analysis was first conducted using the Kaplan-Meier method where statistical significance of differences in the cumulative survival curves between groups was evaluated by the long-rank test. Multivariate analysis was performed using the Cox's proportional hazard method. Concordance between results from both observers was evaluated using Kappa (j) agreement test. Statistical analysis was conducted using the software SPSS for windows, version 14. Statistical analysis was two sided and significance was defined if P \ 0.05.

Results

Distribution of lymphatics in normal breast tissue and in tumours

The distribution of lymphatics in areas of normal breast tissue surrounding the tumour and in the three tumoural areas was examined. In the normal areas, lymphatics were found to be more or less evenly distributed in the interlobular and interlobar areas but completely absent from the intralobular compartment (Fig. 2a). In tumours, the majority of lymphatics were located mainly in the peripheral and peritumoural areas. Peritumoural lymphatics (PT-L) were detected in all 177 tumours while peripheral lymphatics (PP-L) were detected in 152 tumour (85.9%) compared with 73 tumour (41.2%) having intratumoural lymphatics (IT-L), Fig. 2b,c.

IT-LVD ranged from 0.0 to 2.7/mm 2 (median 0.00/mm 2 ), PP-LVD ranged from 0.00 to 3.50/mm 2 (median 0.30/mm 2 ) and PT-LVD ranged from 0.015 to 3.94/mm 2 (median 0.83/mm 2 ) Total-LVD ranged from 0.015 to 8.59/mm 2 (median 1.37/mm 2 ). The means of the IT-LVD, PP-LVD, PT-LVD and total-LVD were 0.3/mm 2 (±0.03), 0.5/mm 2 (±0.04), 1.02/mm 2 (±0.06) and 1.7/mm 2 (±0.1), respectively. Figure 3 illustrates distribution of lymph vessels in the three tumoural areas. By using the median as a cut-off point, 55 (31.1%) tumours were characterized with high LVD.

Relationship between LVD, clinicopathological characteristics and patient prognosis

No association was found between LVD and tumour size or type. Significant association was found between high-grade tumours and higher IT-LVD (P = 0.048). Higher LVD at the intratumoural, peripheral, peritumoural or total LVD were associated with the presence of LN metastasis; P-values were 0.009, \0.001, \0.001, \0.001, \0.001 respectively. Also a strong association between high LVD, poor NPI and advanced tumour stage were detected; the mean LVD was 1.4 ± 0.12, 1.8 ± 0.17 and 2.9 ± 0.38, in good, intermediate and poor prognostic groups respectively (P-value = 0.009). The relationships between high LVD in different tumour areas and other clinicopathological criteria of tumours are summarized in Table 1. Associations indicate that tumours with aggressive features have higher LVDs. Vascular invasion (VI); encompassing both lymphovascular invasion (LVI) and blood vascular invasion (BVI) was assessed in the same specimens and results reported previously [START_REF] Mohammed | Improved methods of detection of lymphovascular invasion demonstrate that it is the predominant method of vascular invasion in breast cancer and has important clinical consequences[END_REF]. Briefly, by using differential expression of blood and lymphatic markers, VI was detected in 56 specimens (31.6%) of which 54 (96.4%) were purely LVI with no BVI. A highly significant association was found between high total LVD and the presence of LVI (P \ 0.001) (Table 2).

As illustrated by the Kaplan-Meier curve (Fig. 4a,b), in univariate analysis of the 132 patients treated with adjuvant therapy, total LVD was significantly associated with shorter overall survival P = 0.015, and DFI P = 0.01. However in multivariate analysis, LVD lost significance when adjusted to tumour grade, size, LN status, and the presence of LVI indicating it is not an independent prognostic factor. Only tumour grade (P = 0.009), LN status (P = 0.015) and LVI (P = 0.022) retained significance in multivariate analysis (Table 3).

Evidence of lymphangiogenesis in breast cancer

MIB-1-positive LECs nuclei were detected in 25 (29%) of specimens (Fig. 2f) with the proliferation fraction (PF-L) ranging from 0 to 2.2%. Figure 4c shows the distribution frequencies of MIB-1-positive LECs in the 85 specimens. When the presence of proliferating LECs was compared between specimens, according to the LVD, no significant association was found; 37.8% of specimens with high LVD had positive MIB-1 LEC nuclei compared with 20% of specimens with low LVD (P = 0.073) (Table 4).

A different morphology of the intratumoural and peripheral lymphatics in comparison with lymphatics in the peritumoural areas was observed. Lymphatics with MIB-1 positive nuclei were characterized with three features; (a) irregular outline with multiple sprouting, (b) more than one endothelial nucleus in the cross section of small lymphatic capillary, which is unusual (Fig. 2h), and (c) the presence of intracytoplasmic lumina within the cytoplasm of the LEC (Fig. 2g). Lymphatics in the peritumoural areas were, in comparison, more regular, had thinner LEC nuclei with the larger vessels invariably negative for MIB-1 (Fig. 2j). Certain lymphatic vessels in the peritumoural area did, however, show MIB-1 positivity i.e. the smaller vessels immediately adjacent to the peripheral tumour area.

Proliferative status of tumour cells within lymphatics

As reported previously [START_REF] Mohammed | Improved methods of detection of lymphovascular invasion demonstrate that it is the predominant method of vascular invasion in breast cancer and has important clinical consequences[END_REF], it appears that, with the current cohort of specimens, breast tumour cells preferentially invade lymphatics rather than blood vessels. It was therefore of interest to assess the proliferative fraction of tumour cells (emboli) within such vessels (PF-E). PF-E was assessed in 47 specimens in whom LVI was still detected after deeper sectioning. From those 47 specimens MIB-1 positive nuclei of invading tumour cells were detected in 40 (93.8%) specimens. In seven (8.2%) specimens, tumour emboli were MIB-1-negative. The PF-E ranged from 0 to 90% with a median of 26% and mean of 32.5 ± 3.9%. Figure 4d shows the distribution frequencies of PF-E in the 47 specimens. To examine whether there was an association between the PF-T and PF-E, a Kruskal-Wallis test was conducted. Tumours which were characterized by high PF-T also had a high PF-E. Twenty-five of the 47 specimens were characterized with high PF-T. Sixteen out of them had high PF-E, P = 0.006 (Table 5).

Relationship between density of tumoural inflammatory infiltrate, LVD and proliferation fraction of lymphatics

Examination of the density of inflammatory cells in tumour sections (stained by the double staining technique) showed that 66 specimens (77%) had mild infiltrate compared with 19 (22%) with dense infiltrate. The presence of dense inflammatory infiltrate was significantly associated with the presence of proliferating LEC where 58% of tumour with dense inflammatory infiltrate had positive MIB-1 LEC nuclei (Fig. 2f) compared with 21% of tumours with mild inflammatory infiltrate, P = 0.002 using Chi squared test (Table 6).

Angiogenic characteristics and relationship with lymphangiogenesis and survival

The relationship between MVD and LVD was of interest for aforementioned reasons. No statistically significant relationship was detected between high MVD and high LVD; certain tumours with high MVDs had low LVDs and vice versa (Fig. 2d,e). Tumours with high MVD were highly significantly associated with larger tumour size (P \ 0.001), higher grade (P \ 0.001), presence of LN status (P \ 0.001) and negativity of hormonal receptors; P = 0.001 for estrogen receptors and P = 0.006 for progesterone receptors (Table 7). There was a trend for patients whose tumours had high MVD to have poorer survival. The 10-years OS was 93% in patients with low MVD compared with 88.2% of patients with high MVD, however this trend did not reach statistical significance (P = 0.316).

Discussion

The topography of lymphatics in malignant tumours, particularly in breast cancer, has, for some time, been a hotly debated issue in the literature. Initial studies investigated lymphatics in xenografted sarcomas and reported that vessels were only found in peritumoural areas and absent from the intratumoural compartment [START_REF] Leu | Absence of functional lymphatics within a murine sarcoma: a molecular and functional evaluation[END_REF][START_REF] Padera | Lymphatic metastasis in the absence of functional intratumor lymphatics[END_REF]. With advances in imaging techniques, intratumoural lymphatics were detected and found to be functional, with tumour cells detected flowing within the vessels [START_REF] Pathak | Visualizing function in the tumor-associated lymphatic system[END_REF]. Controversy exists regarding lymphatics in human breast cancer, with some studies reported absence of intratumoural lymphatics [START_REF] Agarwal | Lymphangiogenesis does not occur in breast cancer[END_REF][START_REF] Vleugel | Lack of lymphangiogenesis during breast carcinogenesis[END_REF][START_REF] Williams | Absence of lymphangiogenesis and intratumoural lymph vessels in human metastatic breast cancer[END_REF] and others reporting their presence [START_REF] Bono | High LYVE-1-positive lymphatic vessel numbers are associated with poor outcome in breast cancer[END_REF][START_REF] Nakamura | Lymph vessel density correlates with nodal status, VEGF-C expression, and prognosis in breast cancer[END_REF]. In the present study, lymphatics were detected in the peritumoural area in all specimens while 86 and 41% of specimens have additional lymphatics in the peripheral and in the inner 2/3 core of the tumour, respectively. The controversy regarding the presence or absence of intratumoural lymphatics may exist for many reasons. Breast cancer is a heterogeneous disease; therefore the patient population will vary considerably from one study to another. A contributory factor to conflicting results is that different terms have been used to describe the location of tumoural lymphatics. In the current study, intratumoural lymphatics referred to vessels within the inner 2/3 core of the tumour including vessels both among the tumour cells and/or in the tumour stroma. Other studies refer to intratumoural lymphatics as any vessels within the tumour area, either in the peripheral part or in the inner core [START_REF] Van Der Auwera | Tumor lymphangiogenesis in inflammatory breast carcinoma: a histomorphometric study[END_REF]. Intratumoural lymphatics have also been referred to as vessels present only among tumour cells, while those present within the tumour stroma were separately counted and analyzed [START_REF] Van Der Schaft | Absence of lymphangiogenesis in ductal breast cancer at the primary tumor site[END_REF]. In a recent study, lymphatics at the peripheral area or the tumour were referred to as lymphatics at the advanced tumour edge while peritumoural lymphatics were referred to as lymphatics in the nontumour area [START_REF] Yamauchi | Accurate assessment of lymph vessel tumor emboli in invasive ductal carcinoma of the breast according to tumor areas, and their prognostic significance[END_REF]. Results from the current study are in concordance with previous studies showing that, in breast cancer, tumoural lymphatics are mainly found in the peritumoural and advancing edge of the tumour. Intratumoural lymphatics are, however, found in a number of patients, seem to be functional, as discussed below, and may have important clinical consequences.

The relationship between LVD and clinicopathological criteria has not been firmly established as yet. The present study, as with others, found a significant positive association between high LVD and the presence of LN metastasis and tumours with negative hormonal receptor status [START_REF] Choi | Angiogenic and lymphangiogenic microvessel density in breast carcinoma: correlation with clinicopathologic parameters and VEGF-family gene expression[END_REF][START_REF] Nakamura | Lymph vessel density correlates with nodal status, VEGF-C expression, and prognosis in breast cancer[END_REF]. Such associations were not found in other studies [START_REF] Kato | A quantitative analysis of lymphatic vessels in human breast cancer, based on LYVE-1 immunoreactivity[END_REF][START_REF] Van Der Schaft | Absence of lymphangiogenesis in ductal breast cancer at the primary tumor site[END_REF]. This may be due to different approaches used to assess LVD where it was assessed by counting the immunostained vessels in selected tumoural hot spots [START_REF] Choi | Angiogenic and lymphangiogenic microvessel density in breast carcinoma: correlation with clinicopathologic parameters and VEGF-family gene expression[END_REF][START_REF] Bono | High LYVE-1-positive lymphatic vessel numbers are associated with poor outcome in breast cancer[END_REF][START_REF] Kato | A quantitative analysis of lymphatic vessels in human breast cancer, based on LYVE-1 immunoreactivity[END_REF][START_REF] Nakamura | Lymph vessel density correlates with nodal status, VEGF-C expression, and prognosis in breast cancer[END_REF] or in randomly selected fields [START_REF] Van Der Schaft | Absence of lymphangiogenesis in ductal breast cancer at the primary tumor site[END_REF].

Although high LVD was significantly associated with shorter OS, the prognostic power of LVD was not strong enough to retain significance when adjusted to other prognostic factors such as tumour grade, LN status, tumour size and LVI. A similar conclusion has been reported both in studies on breast cancer [START_REF] Nakamura | Lymph vessel density correlates with nodal status, VEGF-C expression, and prognosis in breast cancer[END_REF] and in other types of cancers [START_REF] Miyata | Lymphangiogenesis and angiogenesis in bladder cancer: prognostic implications and regulation by vascular endothelial growth factors-A, -C, and -D[END_REF]. Current results suggest that rather than it being the number of the lymph vessels that is important in determining whether tumour cells will metastasise, it is the ability of the tumour cells to invade through the vessel that is of greatest importance. As such it was of interest to examine if these lymphatics were newly formed or were pre-existing. In the study of Van der Auwera et al. [START_REF] Van Der Auwera | Increased angiogenesis and lymphangiogenesis in inflammatory versus noninflammatory breast cancer by real-time reverse transcriptase-PCR gene expression quantification[END_REF]. Ki-67-positive LECs were detected in 16 (*50%) noninflammatory and 12 (80%) inflammatory specimens whereas the study by van der Schaft et al. [START_REF] Van Der Schaft | Absence of lymphangiogenesis in ductal breast cancer at the primary tumor site[END_REF] reported no proliferative LECs (10 specimen were studied). The present study examined 85 specimens and proliferative lymphatics were detected in 25 (29%) with PF-L ranging from 0 to 2.2%. PF-L ranged from 0 to 3% in studies of other tumour types, [START_REF] Soares | Lymphatic vascular density and lymphangiogenesis during tumor progression of carcinoma expleomorphic adenoma[END_REF][START_REF] Stefansson | Vascular proliferation is important for clinical progress of endometrial cancer[END_REF][START_REF] Sipos | Expression of lymphangiogenic factors and evidence of intratumoral lymphangiogenesis in pancreatic endocrine tumors[END_REF]. The low PF-L is undoubtedly due to the inability of the methodology to accurately detect all dividing LECs within tumours, and is most probably underestimated as sprouting of new vessels begins at different levels along the lymphatic vessel [START_REF] Adams | Molecular regulation of angiogenesis and lymphangiogenesis[END_REF]. Furthermore, new vessels may originate from circulating progenitors or from non-endothelial cells via trans-differentiation [START_REF] Maruyama | Inflammation-induced lymphangiogenesis in the cornea arises from CD11b-positive macrophages[END_REF].

Intriguingly the presence of large number of lymphatics in tumour areas which were densely infiltrated by inflammatory cells was noted. This finding is supported by studies investigating the role of macrophages and inflammatory cells in inducing lymphangiogenesis in tumours and other tissues [START_REF] Kerjaschki | The crucial role of macrophages in lymphangiogenesis[END_REF][START_REF] Yano | Glucocorticoids suppress tumor lymphangiogenesis of prostate cancer cells[END_REF], and could be due to macrophages being a source of angio-and lymphangiogenic growth factors [START_REF] Schoppmann | Tumour-associated macrophages express lymphatic endothelial growth factors and are related to peritumoral lymphangiogenesis[END_REF].

The present study also found that most of the tumour cells within lymphatics were actively proliferating and that the PF of these tumour emboli positively correlates with the PF of the surrounding tumour cells. Such findings indicate that, in terms of proliferation, the cell population within lymphatics have a similar phenotype to the rest of the tumour cells outside the vessels supporting the notion that the ability of the tumour cells to invade and to metastasis is not a clonal trait [START_REF] Weigelt | Molecular portraits and 70-gene prognosis signature are preserved throughout the metastatic process of breast cancer[END_REF]. However there was no association between the PF of the tumour and the presence of LVI i.e. tumours with high proliferation do not necessarily invade vessels suggesting that proliferation is not the only determinant of invasiveness.

In concordance with other studies [START_REF] Bono | High LYVE-1-positive lymphatic vessel numbers are associated with poor outcome in breast cancer[END_REF][START_REF] Schoppmann | Lymphatic microvessel density and lymphovascular invasion assessed by anti-podoplanin immunostaining in human breast cancer[END_REF], no association was found between high LVD and high MVD suggesting that although both processes share some common mechanisms of molecular/cellular regulation [START_REF] Mohammed | Prognostic significance of vascular endothelial cell growth factors -A, -C and -D in breast cancer and their relationship with angio-and lymphangiogenesis[END_REF][START_REF] Adams | Molecular regulation of angiogenesis and lymphangiogenesis[END_REF], the tissue regulation can vary significantly from each other. A significant association was found between high MVD and lager tumour size, higher grade, negativity of hormonal receptors, the presence of LN metastasis and advanced tumour stage. Similar results were reported in the study of Hansen et al. [START_REF] Hansen | The prognostic value of angiogenesis by Chalkley counting in a confirmatory study design on 836 breast cancer patients[END_REF], which is the largest study conducted investigating angiogenesis in breast cancer (836 patients). These associations reflect the importance of angiogenesis in tumour progression. However, unlike their results, we could not find a significant association between high MVD and DFI or OS. In an interesting review, and meta-analysis, by Uzzan et al., examining 87 breast cancer angiogenesis studies; 25 reported significant association between MVD and DFI and 11 with OS [START_REF] Uzzan | Microvessel density as a prognostic factor in women with breast cancer: a systematic review of the literature and meta-analysis[END_REF]. The authors concluded that inter-study variation could result from patient selection criteria, staining techniques and methodology used in counting blood vessels and in selection of the cut-off level for MVD assessment. They also stated that MVD is a significant, although weak, prognostic factor in breast cancer and that standardisation of MVD assessment is needed. Recommendations for such are mentioned in the review by Fox and Harris with regards the different methodologies used to assess angiogenesis [START_REF] Fox | Histological quantitation of tumour angiogenesis[END_REF].

In conclusion, the current study shows that, in breast cancer, lymphatics are located primarily in the peritumoural and peripheral areas and seem to play an important role in disease progression by being likely routes for tumour cell dissemination. It should be noted, however, that proliferating intratumoural lymphatics also occur and appear to be functional in that tumour emboli are observed within them. LVI, but not LVD, is an independent prognostic factor. Tumours differ in their lymphangiogenic and angiogenic characteristics indicating two distinctive biological processes. Lymphangiogenesis does occur in breast cancer, albeit in a small proportion of specimens, with inflammatory cell infiltrate seeming to play an important role in its regulation.

Fig. 2

 2 Fig. 2 (a) Normal breast tissue showing the structural unit of the breast; a central duct (arrow) surrounded with multiple lobules (asterisk). Lymphatics are distributed within the interlobular stroma and completely absent from the intralobular stroma, 940 magnification. (b, c) Two infiltrating ductal carcinomas stained with D2-40 showing multiple lymphatics at the peritumoural area (b, 9100) and intratumoural lymphatics loaded with multiple tumour cells (c, 9200). (d, e) infiltrating ductal carcinoma stained with D2-40 and CD34 respectively showing few lymphatics (arrow) in (d) yet high number of blood capillaries in (e), an example of a tumour with low LVD yet high MVD, 940. (f-j) are tumours stained by double IHC with D2-40 and MIB-1. (f) Tumour section with a lymphatic capillary showing a proliferating LEC with a MIB-1-positive nucleus (black arrow) and a

Fig. 3

 3 Fig. 3 Histograms showing distribution of intratumoural LVD (a), peripheral LVD (b), peritumoural LVD (c) and total LVD (d) in the 177 studied breast cancer specimens

Fig. 4

 4 Fig. 4 (a) and (b) are Kaplan-Meier curves showing positive relationship between higher LVD and shorter overall survival (a) (P = 0.015) and disease free interval (P = 0.01) (b). (c) Histogram shows distribution of MIB-1-positive LECs in whole tumour sections. (d) Histogram showing the distribution of proliferation fraction of tumour cells (emboli) within lymphatics (PF-E) with the majority tumour emboli having MIB-1-positive reactivity and the PF-E ranging from 20 to 90%

Table 1

 1 Association between LVD at the three tumour areas and total LVD and clinicopathological criteria

		P-value	\0.001					0.050				0.089				0.124				0.029				0.106				0.315				0.849
		Total	121	43	13	177		56	112	168		72	93	165		136	41	177		156	18	174		51	126	177		65	112	177		45	62	70
		High	19(15)	29(67)	7(54)			23(41)	29(26)			27(38)	23(25)			38(28)	17(41)			44(28)	10(56)			11(22)	44(35)			17(26)	38(34)	55		12(27)	22(35)	21(30)
	Total-LVD	Low	102(85)	14(33)	6(46)			33(59)	83(74)			45(63)	70(75)			98(72)	24(39)			112(72)	8(44)			40(78)	82(65)			48(74)	74(66)	122		33(73)	40(65)	49(70)
		P-value	\0.001					0.191				0.435				1.000				0.320				0.868				0.539				0.634
		Total	121	43	13	177		56	112	168		72	93	165		136	41	177		156	18	174		51	126	177		65	112	177		45	62	70	177
		High	47(39)	29(67)	11(85)			32(57)	51(45)			38(53)	43(46)			67(49)	20(49)			75(48)	11(61)			26(51)	61(48)			34(52)	53(47)	87		22(49)	33(53)	32(46)
	PT-LVD	Low	73(61)	14(33)	2(15)			24(43)	61(55)			34(47)	50(54)			69(51)	21(51)			81(52)	7(39)			25(49)	65(52)			31(48)	59(53)	90		23(51)	29(47)	38(54)
		P-value	0.001					0.049				0.638				0.304				0.081				0.621				0.087				0.077
		Total	121	43	13	177		56	112	168		72	93	165		136	41	177		156	18	174		51	126	177		65	112	177		45	62	70	177
		High	46(38)	35(81)	8(61)			35(62)	51(45)			39(54)	46(49)			65(48)	24(59)			76(48)	13(72)			24(47)	65(52)			27(42)	62(55)	89		22(49)	25(40)	42(60)
	PP-LVD	Low	75(62)	8(18)	5(39)			21(38)	61(55)			33(46)	47(51)			71(52)	17(41)			80(51)	5(28)			27(53)	61(48)			38(58)	50(45)	88		23(51)	37(60)	28(40)
		P-value	0.005					0.003				0.028				0.151				0.464				0.238				0.321				0.048
		Total	121	43	13	177		56	112	168		72	93	165		136	41	177		156	18	174		51	126	177		65	112	177		45	62	70	177
		High	40(33)	26(61)	75(4)			33(59)	38(34)			38(52)	33(36)			52(38)	21(51)			64(41)	9(50)			25(49)	48(38)			20(31)	53(47)			17(38)	19(31)	37(53)
	IT-LVD	Low	81(67)	17(39)	64(6)			23(41)	74(66)			34(47)	60(65)			84(62)	20(49)			92(59)	9(50)			26(51)	78(62)			45(69)	59(53)			28(62)	43(69)	33(47)
		Stage	I	II	III	Total	ER	Negative	Positive	Total	PR	Negative	Positive	Total	RR	No	Definite	Total	DM	Yes	No	Total	Age	B50 years	[50 years	Total	Size	B1.5 cm	[1.5 cm	Total	Grade	I	II	III	Total

Table 2

 2 Association between LVD and LVI

	LVD	LVI (%)			P-value
		Absent	Present	Total
	Low	109(89)	13(11)	122	\0.001
	High	14(24)	41(75)	55
		123	54	177

Table 3

 3 Multivariate analysis of LVD and MVD adjusted to other prognostic factors

						Sig.	Exp(B)	95.0% CI for Exp(B)
						Lower	Upper
			Size (B vs. [1.5 cm)		0.891	0.888	0.162	4.865
			Grade (I vs. II & III)		0.009	4.423	1.771	19.137
			LVD status (high versus low)	0.604	0.603	0.089	4.070
			MVD status (high versus low)	0.346	0.550	0.159	1.906
			LVI (negative versus positive)	0.022	4.157	1.352	18.218
			LN status (negative versus positive)	0.015	4.829	1.222	19.057
	Significant P-values are bold
	Table 4 Association between LVD and proliferation of tumoural
	lymphatics
	LVD	MIB-1-Negative	MIB-1-positive	Total	P-value
	status	LEC nuclei	LEC nuclei
	Low	32 (80%)	8 (20%)	40 (47.1%) 0.07
	High	28 (62.2%)	17 (37.8%)	45 (52.9%)
		60	25	85

Table 5

 5 Association between proliferation fraction of the tumour emboli within lymphatics (PF-E) and the PF of tumour cells outside the vessels (PF-T)

	PF of tumour emboli PF of the tumour	Total P-value
		ells outside the vessels
		High	Low
	High	16	6	22	0.006
	Low	7	18	25
	Total	23	24	47

Table 6

 6 Association between density of inflammatory infiltrate within the tumour and proliferation of tumoural lymphatics

	Density of inflam-	MIB-1-Negative	MIB-1-posi-	Total P-value
	matory infiltrate	LEC nuclei	tive LEC
			nuclei
	Low	52 (79%)	14 (21%)	66	0.002
	High	8 (42%)	11 (58%)	19
	Total	60	25	85

Table 7

 7 Association between MVD and clinicopathological criteria

	Breast Cancer Res Treat (2009) 113:261-273
	MVD (%)
		Low	High	Total	P-value		Low	High	Total	P-value
	Age					ER
	\50 years	26(51)	25(49)	51	0.383	Negative	20(36)	36(64)	56	0.001
	[50 years	75(49)	51(41)	126		Positive	72(64)	40(36)	112
				177					168
	Size					PR
	\1.5 cm	81(79)	14(21)	65	\0.0001	Negative	30(42)	42(58)	72	0.006
	[1.5 cm	50(45)	62(55)	112		Positive	60(65)	33(35)	93
				177					165
	Grade					NPI
	I, II	75(70)	32(30)	107	\0.0001	Good	62(83)	12(16)	74	\0.0001
	III	26(37)	44(63)	70		Intermediate	32(36)	56(63)	88
				177		Poor	7(46)	8(53)	15
	LN status					RR
	Negative	82(66)	43(34)	125	\0.0001	No	79(58)	57(42)	136	0.880
	Positive	19(37)	33(63)	52		Definite	22(54)	19(46)	41
				177					177
	Stage					LVD
	I	79(65)	42(35)	121	0.001	Low	74(61)	48(39)	122	0.189
	II	17(40)	26(60)	43		High	27(49)	28(51)	55
	III	5(38)	8(62)	13					177
				177
	ER-estrogen receptor, PR-progesterone receptor, NPI-Nottingham prognostic index, RR-regional recurrence
	Significant P-values are bold

Breast Cancer Res Treat (2009) 113:261-273

Acknowledgments Rabab A. A. Mohammed is very grateful to the Egyptian government for funding her during this work. The authors are also grateful to Mrs. Kelly Huber and Miss Aula Ammar (University of Nottingham, University Hospitals, Department of Clinical Oncology) for their kind technical support and valuable advice.


Daniela Bonofiglio

Sabrina Gabriele

Saveria Aquila

Hongyan Qi

Maria Belmonte

Stefania Catalano

Sebastiano Andò

A E Sabrina

Gabriele Ae

A E Hongyan

Qi Ae

A E Stefania

Catalano Ae

Sebastiano Ando
email: sebastiano.ando@unical.it

Á S Gabriele

Á S Aquila

Á H Qi

Á M Belmonte

Á S Catalano

Á S Ando

Peroxisome proliferator-activated receptor gamma activates fas ligand gene promoter inducing apoptosis in human breast cancer cells

Keywords: PPARc, FasL, Apoptosis, Caspase 8, Breast cancer

come

Introduction

Since its discovery at previous decade, it became clear that peroxisome proliferator-activated receptor gamma (PPARc) is an important modulator in the regulation of complex pathways of mammalian cells' metabolism and recently implicated in cancer [START_REF] Francis | Nuclear receptors and the control of metabolism[END_REF][START_REF] Shearer | Recent advances in peroxisome proliferator-activated receptor science[END_REF][START_REF] Kota | An overview on biological mechanisms of PPARs[END_REF]. Several antineoplastic effects, such as induction of apoptosis and differentiation, have been described as a result of PPARc-mediated action both in vitro and in vivo [START_REF] Elstner | Ligands for peroxisome proliferator-activated receptor gamma and retinoic acid receptor inhibit growth and induce apoptosis of human breast cancer cells in vitro and in BNX mice[END_REF][START_REF] Haydon | Nuclear receptor agonists as potential differentiation therapy agents for human osteosarcoma[END_REF][START_REF] Konopleva | Role of peroxisome proliferator-activated receptor-gamma in hematologic malignancies[END_REF][START_REF] Panigrahy | Therapeutic potential of thiazolidinediones as anticancer agents[END_REF][START_REF] Suzuki | Peroxisome proliferatoractivated receptor gamma in human breast carcinoma: a modulator of estrogenic actions[END_REF][START_REF] Yu | Troglitazone inhibits tumor growth in hepatocellular carcinoma in vitro and in vivo[END_REF][START_REF] Kim | Activation of peroxisome proliferator-activated receptorgamma (PPARgamma) induces cell death through MAPKdependent mechanism in osteoblastic cells[END_REF], although the molecular mechanisms by which PPARc causes these effects remain to be fully elucidated. PPARc is able to be activated by ligands of physiological or pharmacological origin [START_REF] Lehrke | The many faces of PPARgamma[END_REF]. Synthetic PPARc(ligands are the thiazolidinediones (TZDs), a class of oral antidiabetic agents, which exert anti-tumoral effects depending on the experimental and cellular settings and in a PPARc-dependent or PPARc-independent fashion [START_REF] Grommes | Antineoplastic effects of peroxisome proliferator-activated receptor gamma agonists[END_REF][START_REF] Turturro | Troglitazone and pioglitazone interactions via PPAR{-gamma} independent and dependent pathways in regulating physiological responses in renal tubule derived cell lines[END_REF].

In mammals, apoptosis can be initiated by two major routes: the intrinsic and extrinsic death pathways [START_REF] Green | Apoptotic pathways: the roads to ruin[END_REF]. The instrinsic pathway is triggered in response to a variety of apoptotic stimuli that produce damage within the cell, including anticancer agents, oxidative damage, UV irradiation, and is mediated through the mitochondria. The extrinsic pathway is activated by extracellular ligands able to induce oligomerization of death receptors, such as Fas (also called CD95 or APO1) or other members of the tumor necrosis factor receptor superfamily leading to apoptosis [START_REF] Nagata | Apoptosis by death factor[END_REF].

Fas ligand (FasL), a type II transmembrane protein expressed on the surface of cells, induces apoptotic cell death by binding to its receptor Fas [START_REF] Chinnaiyan | Portrait of an executioner: the molecular mechanism of FAS/APO-1-induced apoptosis[END_REF][START_REF] Debatin | Apoptosis pathways in cancer, cancer therapy[END_REF]. This binding results in recruitment of the Fas-associated death domain (FADD) protein and caspase 8 zymogens to the receptor and the formation of the death-inducing signalling complex, after which the caspases cascade can be activated [START_REF] Pinkoski | Fas ligand, death gene[END_REF]. Defects in the Fas/FasL apoptotic signalling pathway provide a survival advantage to cancer cells and may be implicated in tumorigenesis [START_REF] Wajant | CD95L/FasL and TRAIL in tumour surveillance and cancer therapy[END_REF]. Recent evidence has indicated the Fas pathway in the induction of tumor cell death and FasL expression has been observed in different tumors including breast cancer [START_REF] O'connell | The Fas counterattack: Fas-mediated T cell killing by colon cancer cells expressing Fas ligand[END_REF][START_REF] Muschen | CD95 ligand expression in dedifferentiated breast cancer[END_REF][START_REF] Reimer | FasL:Fas ratio-a prognostic factor in breast carcinomas[END_REF]. It has also been suggested that FasL functions as an autocrine/paracrine mediator of apoptosis induced by DNA-damaging anticancer chemotherapeutic agents [START_REF] Kasibhatla | DNA damaging agents induce expression of Fas ligand and subsequent apoptosis in T lymphocytes via the activation of NF-kappa B and AP-1[END_REF][START_REF] Mo | DNA damage signals induction of fas ligand in tumor cells[END_REF].

In the past few years, we have investigated different molecular mechanisms through which PPARc induces antiproliferative effects, cell cycle arrest and apoptosis in human MCF7 breast cancer cells [START_REF] Bonofiglio | Estrogen receptor alpha binds to peroxisome proliferator-activated receptor (PPAR) response element and negatively interferes with PPAR gamma signalling in breast cancer cells[END_REF][START_REF] Bonofiglio | Peroxisome proliferator-activated receptor-gamma activates p53 gene promoter binding to the nuclear factor-kappaB sequence in human MCF7 breast cancer cells[END_REF]. In this study, we reported for the first time that PPARc directly activates FasL promoter gene inducing apoptotic events in breast cancer cells.

Materials and methods

Cell cultures

Human MCF7 (a gift from Dr. Ewa Surmacz, Sbarro Institute for Cancer Research and Molecular Medicine, Philadelphia, USA) MDA-MB231 (ERa-negative, MDA) breast cancer cells were grown in DMEM plus glutamax containing 10% fetal calf serum (FCS) (Invitrogen, Milan, Italy) and 1 mg/ml penicillin-streptomycin (P/S). BT20 (insulin receptor substrate-1, IRS-1 negative) breast cancer cells were grown in MEM added as DMEM.

Reagents

Rosiglitazone (BRL49653, BRL) was from Alexis (San Diego, CA USA), the irreversible PPARc-antagonist GW and the Sp1 specific inhibitor, mithramycin were from Sigma (Milan, Italy).

Plasmids

The firefly luciferase reporter plasmids containing FasL promoter wild-type (FasL Luc-0) or its deletion mutants (FasL Luc-3 and FasL Luc-4) were kind gifts from Dr. Carlos V. Paya (Mayo Clinic, Rochester, Minnesota, USA). Deletion of Sp1 sequence in FasL gene promoter was generated by PCR using as template FasL Luc-3 (p-318 construct). The resulting plasmid encoding the human FasL gene promoter containing the desired deletion was designed FasL Luc-3 DSp1 and the sequence was confirmed by nucleotide sequence analysis [START_REF] Catalano | Fas ligand expression in TM4 sertoli cells is enhanced by estradiol ''in situ'' production[END_REF]. As an internal transfection control, we co-transfected the plasmid pRL-CMV (Promega Corp., Milan, Italy) that expresses Renilla luciferase enzymatically distinguishable from firefly luciferase by the strong cytomegalovirus enhancer/promoter.

Immunoblotting

Cells were grown in 10 cm dishes to 70-80% confluence and exposed to treatments in serum free medium (SFM) as indicated. Cells were then harvested in cold phosphatebuffered saline (PBS) and resuspended in lysis buffer containing 20 mM HEPES pH 8, 0.1 mM EDTA, 5 mM MgCl 2 , 0.5 M NaCl, 20% glycerol, 1% NP-40, inhibitors (0.1 mM Na 3 VO 4 , 1% PMSF, 20 mg/ml aprotinin). Protein concentration was determined by Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA USA). A 60 lg portion of protein lysates was used for WB resolved on a 12% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane and probed with antibodies (Abs) directed against the FAS, FasL and PPARc (Santa Cruz Biotechnology, CA USA), caspase 8 (Biomol International, Butler PikePlymouth, PA USA). As internal control, all membranes were subsequently stripped (glycine 0.2 M, pH 2.6 for 30 min at room temperature) of the first antibody and reprobed with anti-b-actin Ab (Santa Cruz Biotechnology).

The antigen-antibody complex was detected by incubation of the membranes for 1 h at room temperature with the appropriated secondary antibodies peroxidase-coupled and revealed using the enhanced chemiluminescence system (ECL system, Amersham Pharmacia, Buckinghamshire, UK). Blots were then exposed to film (Kodak film, Sigma). The intensity of bands representing relevant proteins was measured by Scion Image laser densitometry scanning program.

Reverse transcription-polymerase chain reaction (RT-PCR) assay MCF7 cells were grown in 10 cm dishes to 70-80% confluence and exposed to treatments for 24 h in SFM. Total cellular RNA was extracted using TRIZOL reagent (Invitrogen) as suggested by the manufacturer. The purity and integrity were checked spectroscopically and by gel electrophoresis before carrying out the analytical procedures. The evaluation of gene expression was performed by semiquantitative RT-PCR method as previously described [START_REF] Maggiolini | Non-radioactive method for inexpensive quantitative RT-PCR[END_REF]. For FasL and the internal control gene 36B4, the primers were: 5 0 -GGA ATG GGA AGA CAC CTA TGG A-3 0 (FasL forward) and 5 0 -AGA GAG AGC TCA GAT ACG TTG AC-3 0 (FasL reverse), 5 0 -CTC AAC ATC TCC CCC TTC TC-3 0 (36B4 forward) and 5 0 -CAA ATC CCA TAT CCT CGT CC-3 0 (36B4 reverse) to yield respectively products of 299 bp with 25 cycles and 408 bp with 12 cycles. The results obtained as optical density arbitrary values were transformed to percentage of the control (percent control) taking the samples from untreated cells as 100%.

Transient transfection assay MCF7 cells were transferred into 24 well plates with 500 ll of regular growth medium/well the day before transfection. The medium was replaced with SFM on the day of transfection, which was performed using Fugene 6 reagent as recommended by the manufacturer (Roche Diagnostics, Mannheim, Germany) with a mixture containing 0.5 lg of promoter-luciferase reporter plasmid and 5 ng of pRL-CMV. After 24 h transfection, treatments were added in SFM as indicated and cells were incubated for further 24 h, cells were pre-treated for 2 h with mithramycin or GW where necessary. Firefly and Renilla luciferase activities were measured using the Dual Luciferase Kit (Promega). The firefly luciferase values of each sample were normalized by Renilla luciferase activity and data were reported as Relative Light Units (RLU) values.

Electrophoretic mobility shift assay (EMSA)

Nuclear extracts from MCF7 cells were prepared as previously described [START_REF] Andrews | A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells[END_REF]. Briefly, MCF7 cells plated into 10 cm dishes were grown to 70-80% confluence shifted to SFM for 24 h and then treated with 1 lM BRL for 6 h. Thereafter, cells were scraped into 1.5 ml of cold PBS. Cells were pelleted for 10 s and resuspended in 400 ll cold buffer A (10 mM HEPES-KOH pH 7.9 at 4°C, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM PMSF, 1 mM leupeptin) by flicking the tube. The cells were allowed to swell on ice for 10 min and then vortexed for 10 s. Samples were centrifuged for 10 s and the supernatant fraction discarded. The pellet was resuspended in 50 ll of cold Buffer B (20 mM HEPES-OH pH 7.9, 25% glycerol, 1.5 mM MgCl 2 , 420 mM NaCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM PMSF, 1 mM leupeptin) and incubated in ice for 20 min for high-salt extraction. Cellular debris were removed by centrifugation for 2 min at 4°C and the supernatant fraction (containing DNA binding proteins) was stored at -70°C. The probe was generated by annealing single stranded oligonucleotides and labeled with [c 32 P] ATP (Amersham Pharmacia) and T4 polynucleotide kinase (Promega) and then purified using Sephadex G50 spin columns (Amersham Pharmacia). The DNA sequence, containing the Sp1, NFAT and NFkB sites, obtained from the native FasL promoter gene used as probe or as cold competitor is the following: 5 0 -A AAT TGT GGG CGG AAA CTT CCA GGG-3 0 (Sigma). As cold competitor we also used DSp1 oligonucleotide: 5 0 -AT TGT GTT CGG AAA CTT CCA GGG-3 0 , DNFAT oligonucleotide: 5 0 -A AAT TGT GGG CGG TCA CT T CCA GGG-3 0 and DNFkB oligonucleotide: 5 0 -A AAT TGT GGG CGG AAA CAT ATA GGG-3 0 (Sigma). The protein binding reactions were carried out in 20 ll of buffer [20 mM Hepes pH 8, 1 mM EDTA, 50 mM KCl, 10 mM DTT, 10% glicerol, 1 mg/ml BSA, 50 lg/ml poly dI/dC] with 50,000 cpm of labeled probe, 5 lg of MCF7 nuclear protein and 5 lg of poly (dI-dC). The mixtures were incubated at room temperature for 20 min in the presence or absence of unlabeled competitors oligonucleotides. For in vitro mithramycin treatment, 100 nM mithramycin were incubated with the labelled probe for 30 min at 4°C before the addition of nuclear extract. For the experiments involving the anti-PPARc and anti-Sp1 Abs (Santa Cruz Biotechnology), the reaction mixture was incubated with Ab at 4°C for 30 min before addition of labeled probe. The entire reaction mixture was electrophoresed through a 6% polyacrylamide gel in 0.25 9 Tris borate-EDTA for 3 h at 150 V. Gel was dried and subjected to autoradiography at -70°C.

Chromatin immunoprecipitation (ChIP) and Re-ChIP assays

For ChIP assay, MCF7 cells were grown in 10 cm dishes to 50-60% confluence, shifted to SFM for 24 h and then treated with 1 lM BRL for 1 h or pre-incubated with mithramycin for 1 h where required. Thereafter, cells were washed twice with PBS and crosslinked with 1% formaldehyde at 37°C for 10 min. Next, cells were washed twice with PBS at 4°C, collected and resuspended in 200 ll of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1) and left on ice for 10 min. Then, cells were sonicated four times for 10 s at 30% of maximal power (Sonics, Vibra Cell 500 W) and collected by centrifugation at 4°C for 10 min at 14,000 rpm. The supernatants were diluted in 1.3 ml of IP buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, 16.7 mM NaCl) followed by immunoclearing with 80 ll of sonicated salmon sperm DNA/protein A agarose (UBI, DBA Srl, Milan-taly) for 1 h at 4°C. The precleared chromatin was immunoprecipitated with anti-PPARc Ab and re-immunoprecipitated with anti-RNA Pol II Ab (Santa Cruz Biotechnology). At this point, 60 ll salmon sperm DNA/ protein A agarose were added and precipitation was further continued for 2 h at 4°C. After pelleting, precipitates were washed sequentially for 5 min with the following buffers: Wash A (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl), Wash B (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl), and Wash C (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.1), and then twice with TE buffer (10 mM Tris, 1 mM EDTA). The immunocomplexes were eluted with elution buffer (1% SDS, 0.1 M NaHCO 3 ). The eluates were reverse crosslinked by heating at 65°C and digested with proteinase K (0.5 mg/ml) at 45°C for 1 h. DNA was obtained by phenol/chloroform/isoamyl alcohol extraction. 2 ll of 10 mg/ml yeast tRNA (Sigma) were added to each sample and DNA was precipitated with 70% ethanol for 24 h at -20°C, and then washed with 95% ethanol and resuspended in 20 ll of TE buffer. A 5 ll volume of each sample was used for PCR amplification with the following primers flanking a sequence of FasL promoter: 5 0 -TAC CCC CAT GCT GAC CTG CTC-3 0 (forward) and 5 0 -ACG GGA CCC TGT TGC TGA CTG-3 0 (reverse) corresponding to the -396 to -76 region (Gene Bank, accession number: AF027385). The PCR conditions were 45 s at 94°C, 40 s at 58°C, and 90 s at 72°C. The amplification products obtained in 30 cycles were analysed in a 2% agarose gel and visualized by ethidium bromide staining. The negative control was provided by PCR amplification without DNA sample. The specificity of reactions was ensured using normal mouse and rabbit IgG (Santa Cruz Biotechnology).

ChIP/immunoblot assay

For ChIP/Immunoblot assay, MCF7 cells were subjected to the procedure of ChIP assay, as above described, until we obtained the immunocomplexes. At this time, we added the Laemmli buffer to the immunocomplexes and performed the WB analysis, as described by the Upstate protocol. The specificity of reactions was ensured using normal rabbit IgG (Santa Cruz Biotechnology).

RNA interference (RNAi)

Cells were plated in 6 well dishes with regular growth medium the day before transfection to 60-70% confluence. On the second day the medium was changed with SFM without P/S and cells were transfected with 25 bp RNA duplex of stealth RNAi targeted human FasL mRNA sequence 5 0 -GCC CAU UUA ACA GGC AAG UCC AAC U-3 0 (Invitrogen), with 25 bp RNA duplex of validate RNAi targeted human PPARc mRNA sequence 5 0 -GCC UGC AUC UCC ACC UUA UUA UUC U-3 0 or with a stealth RNAi control (Invitrogen) to a final concentration of 100 nM using Lipofectamine 2000 (Invitrogen) as recommended by the manufacturer. After 5 h the transfection medium was changed with SFM in order to avoid Lipofectamine 2000 toxicity and cells were exposed to 1 lM BRL for the next 48 h and then lysed as described for WB analysis or treated for 72 h and then collected as described for the DNA fragmentation.

DNA fragmentation

DNA fragmentation was determined by gel electrophoresis. Cells were grown in 10-cm dishes to 70% confluence, PPARc or control RNAis and then treated with 1 lM BRL and/or 10 lM GW. After 72 h cells were collected and washed with PBS and pelletted at 1,800 rpm for 5 min. The samples were resuspended in 0.5 ml of extraction buffer (50 mM, Tris-HCl, pH 8; 10 mM EDTA, 0.5% SDS) for 20 min in rotation at 4°C. DNA was extracted with phenolchloroform three times and once with chloroform. The aqueous phase was used to precipitate acids nucleic with 0.1 volumes or of 3 M sodium acetate and 2.5 volumes cold EtOH overnight at -20°C. The DNA pellet was resuspended in 15 ll of H 2 O treated with RNAse A for 30 min at 37°C. The absorbance of the DNA solution at 260 and 280 nm was determined by spectrophotometry. The extracted DNA (40 lg/lane) was subjected to electrophoresis on 1.5% agarose gels. The gels were stained with ethidium bromide and then photographed.

Statistical analysis

Statistical analysis was performed using ANOVA followed by Newman-Keuls testing to determine differences in means. P \ 0.05 was considered as statistically significant.

Results

MCF7 breast cancer cells express both Fas and FasL

Previous studies have shown that MCF7 cells coexpress both Fas and FasL, indeed controversial data are reported in this context [START_REF] Beidler | The baculovirus p35 protein inhibits Fas-and tumor necrosis factor-induced apoptosis[END_REF][START_REF] Keane | Fas expression and function in normal and malignant breast cell lines[END_REF][START_REF] Toillon | Normal breast epithelial cells induce apoptosis of breast cancer cells via Fas signaling[END_REF]. To evaluate if Fas and FasL are expressed in our MCF7 cells, we did WB analysis of cellular lysates and found that they do indeed express both proteins (Fig. 1a).

BRL up-regulates FasL expression in MCF7 cells

In order to understand whether PPARc triggers apoptotic events through Fas/FasL signalling, we first evaluated the ability of PPARc agonist BRL to modulate FasL expression in MCF7 cells. A BRL dose-dependent increase in FasL content was observed by WB after 24 h of treatment (Fig. 1b). Furthermore, by a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) method, after 24 h upon increasing BRL concentration, we showed that BRL was able to upregulate FasL gene expression in a dose-dependent manner (Fig. 1c). The BRL action on FasL espression was reversed when we used GW9662 (GW), a specific PPARc antagonist, (Fig. 1b andc) implying a PPARc-dependent action in MCF7 cells.

PPARc transactivates FasL promoter in MCF7 cells

Next, we evaluated whether one of the mechanisms involved in PPARc-mediated increase of FasL expression could be a direct modulation of the transcriptional activity of its promoter. Transient transfection assays were performed in MCF7 cells using the luciferase reporter constructs containing the region of the human FasL promoter gene spanning from -2365 to -2 bp relative to the translation initiation site (FasL Luc-0) or progressive 5 0 deletions of the FasL promoter, FasL Luc-3 (from -318 to -2 bp) and FasL Luc-4 (from -237 to -2 bp) (Fig. 2a). Of interest, differences in the basal transcriptional activity were observed among constructs. The results shown in Fig. 2b indicate that transcription from the FasL Luc-0 construct was detectable, but strong and increasing luciferase activity was evidenced with FasL Luc-3 and FasL Luc-4 constructs, suggesting the presence of potential negative regulatory regions located within -2365 and -318 bp, consistent with a previous study [START_REF] Mcclure | Constitutive Fas ligand gene transcription in Sertoli cells is regulated by Sp1[END_REF].

Using the FasL Luc-3 construct, the promoter activity increased upon BRL treatment in a dose-dependent manner, while this effect was abrogated by GW, indicating that FasL promoter activation depends on the presence of a functional PPARc (Fig. 2c). BRL failed to enhance the transcriptional activity when cells were transfected with FasL Luc-4 construct (Fig. 2c), suggesting that the segment located between -318 and -237 relative to the translation initiation site is crucial for BRL-responsiveness. In particular, this region contains three DNA motifs known to bind the transcription factors Sp1 (named according to the original purification scheme that included Sephacryl and phosphocellulose columns) [START_REF] Philipsen | A tale of three fingers: the family of mammalian Sp/XKLF transcription factors[END_REF], nuclear factor of activated T cells (NFAT) and nuclear factor-kB (NFkB) (Fig. 2a) [START_REF] Mcclure | Constitutive Fas ligand gene transcription in Sertoli cells is regulated by Sp1[END_REF]. It was reported that Sp1 consensus sequence is necessary for basal transcription from the FasL promoter, and that Sp1 alone is sufficient to drive transcription from the promoter in vivo [START_REF] Mcclure | Constitutive Fas ligand gene transcription in Sertoli cells is regulated by Sp1[END_REF]. We sought to investigate whether Sp1 alone was sufficient to drive FasL transcription or if it required the presence of additional ones. By using a FasL Luc-3 construct deleted in the Sp1 site (Fas Luc-3 DSp1), as shown in Fig. 2a, BRL was unable to transactivate FasL promoter (Fig. 2c). Mithramycin is a drug able to bind to GC boxes and then to inhibit Sp1 binding selectively blocking mRNA synthesis from genes that contain functional recognition sites both in vitro and in vivo [START_REF] Blume | Mithramycin inhibits SP1 binding and selectively inhibits transcriptional activity of the dihydrofolate reductase gene in vitro and in vivo[END_REF]. Besides, as shown in Fig. 2d, in MCF7 cells transfected with FasL Luc-3 construct and treated with mithramycin, the FasL transactivation upon 1 lM BRL treatment was prevented. Altogether these data strongly suggest that Sp1 plays an important role in regulating FasL transcription also in MCF7 cells.

PPARc interacts with FasL through Sp1 site in electrophoretic mobility shift assay (EMSA)

To gain further insight into the mechanism involved in the FasL transactivation induced by BRL, we performed EMSA using a synthetic oligodeoxyribonucleotide which contains the Sp1, NFAT and NFkB DNA binding motifs and encompasses the DNA fragment of FasL promoter from -288 to -263 relative to the translation initiation site. We tested for possible interactions of PPARc with In nuclear extracts from MCF7 cells we observed the formation of three specific complexes (Fig. 3, lane 1) which were increased upon BRL treatment (lane 2). The binding was abrogated by 100 fold molar excess of unlabelled probe (lane 3), demonstrating the specifity of the DNA-binding complex. To confirm that FasL transcription induced by BRL in MCF7 cells is regulated by Sp1, nuclear extracts were incubated with three different unlabelled oligonucleotides bearing internal mutations as reported in Materials and Methods.

An excess of unlabeled oligonucleotide containing mutations in the Sp1 site did not compete with the DNA binding of the protein complex (lane 4). In addition, incubation with an excess of unlabeled oligonucleotides containing mutations of either NFAT or NFkB competed both away all DNA-binding activity of the protein complexes bound to the 32 P-labeled wild type -288 to -263 FasL oligonucleotide (lane 5 and 6, respectively). Moreover, the addition of mithramycin in the reaction mixture strongly reduced the intensity of the bands (Fig. 3, lane 7). The involvement of PPARc and Sp1 in the DNA-binding complexes was confirmed by using the specific anti-PPARc (lane 8) and anti-Sp1 Abs (lane 9), since both induced supershift and immunodepletion of the bands.

Functional interaction of PPARc with FasL by chromatin immuno-precipitation (ChIP) assay

The interaction of PPARc with the FasL gene promoter was further investigated by ChIP assay. MCF7 chromatin was immunoprecipitated with the anti-PPARc Ab and then reprecipitated with the anti-RNA-Pol II or anti-Sp1 Abs. PCR was used to determine the recruitment of PPARc to the FasL region containing the Sp1 site. The results indicated that PPARc was weakly constitutively bound to the FasL promoter in untreated cells and this recruitment was increased upon BRL treatment, while mithramycin combined with BRL reversed this effect (Fig. 4a). In addition, by Re-ChIP assays an increased association of Sp1 was obtained and particularly the augmented RNA-Pol II recruitment indicated that a positive regulation of FasL transcription activity was induced by BRL (Fig. 4a). The physical interaction between PPARc and Sp1 proteins was strengthened by the ChIP/Immunoblot assay (Fig. 4b).

PPARc activates the Fas/FasL apoptotic pathway

Fas/FasL signalling when activated recruits adapter proteins and cysteine proteases such as caspase 8 leading to apoptosis [START_REF] Green | Apoptotic pathways: the roads to ruin[END_REF]. To better define the action of PPARc on Fas/FasL pathway, we used GW as well as both PPARc and FasL RNA interferences (i) to evaluate the activation of caspase 8, key component of the extrinsic apoptotic process. By WB analysis our data showed that untreated MCF7 cells expressed the pro-form of caspase 8, while only after BRL exposure caspase 8 was activated as evidenced by the presence of its 11 kDa cleavage product (Fig. 5c). The active caspase 8 cleavage was absent in cells treated with GW alone or combined with BRL, or inhibiting both the expression of PPARc and FasL by the respective RNAis (Fig. 5c). As shown in Fig. 5a andb, PPARc and FasL RNAis were able to inhibit the two proteins expression respectively. These data demonstrated for the first time that PPARc triggers the apoptotic events via Fas/FasL signalling, showing that the extrinsic death ). An excess of three unlabeled oligonucleotides DSp1 (lane 4), DNFAT (lane 5) and DNFkB (lane 6), was used. Anti-PPARc and anti-Sp1 Abs were incubated with nuclear extracts from MCF7 cells treated with 1 lM BRL (lanes 8 and 9, respectively). Lane 10 contains probe alone pathway is a significant contributor in BRL-induced apoptosis in MCF7 cells.

The apoptotic process is associated with morphological changes and biochemical events such as nuclear condensation and fragmentation, the fragments correspond to strands of DNA that were cleaved at internucleosomal regions and create a 'ladder pattern' when electrophoresed on an agarose gel [START_REF] Montague | Cellular catabolism in apoptosis: DNA degradation and endonuclease activation[END_REF]. Because of its near universality, internucleosomal DNA degradation is considered a diagnostic hallmark of cells undergoing apoptosis. Therefore, we studied DNA fragmentation assay under BRL treatment in MCF7 cells evidencing that the induced apoptosis is PPARc-dependent as it was reversed by the PPARc RNAi (Fig. 5d). These results indicate a positive crosstalk between PPARc and FasL that is responsible, at least in part, for BRL-induced apoptosis in MCF7 cells.

FasL is a common mechanism by which PPARc mediates apoptosis in breast cancer cells Finally, we examined other human breast cancer cell lines to determine whether the involvement of FasL is unique to MCF-7 cells or it is a common mechanism by which PPARc mediates apoptosis in breast cancer. MDA and BT20 cells, both expressing Fas and FasL (Fig. 6a), showed a FasL upregulation upon BRL treatment, which was reduced by PPARc RNAi (Fig. 6b). DNA fragmentation assay under BRL treatment in MDA and BT20 cells confirmed that the PPARc-induced apoptosis involves Fas/FasL signalling (Fig. 6c), addressing a general mechanism in breast cancer cells.

Discussion

PPARc ligands are shown to be generally antiproliferative and to induce apoptosis in several malignant cell lineages [START_REF] Elstner | Ligands for peroxisome proliferator-activated receptor gamma and retinoic acid receptor inhibit growth and induce apoptosis of human breast cancer cells in vitro and in BNX mice[END_REF][START_REF] Konopleva | Role of peroxisome proliferator-activated receptor-gamma in hematologic malignancies[END_REF][START_REF] Panigrahy | Therapeutic potential of thiazolidinediones as anticancer agents[END_REF][START_REF] Yu | Troglitazone inhibits tumor growth in hepatocellular carcinoma in vitro and in vivo[END_REF]. Additionally, PPARc is proved to be a regulator of the expression of many genes relevant to inhibit carcinogenesis [START_REF] Theocharisa | Peroxisome proliferator-activated receptor-gamma ligands as cell-cycle modulators[END_REF]. Our previous studies evidenced that PPARc, through different pathways, is involved in BRLinduced growth arrest and apoptosis in MCF7 breast cancer cells [START_REF] Bonofiglio | Estrogen receptor alpha binds to peroxisome proliferator-activated receptor (PPAR) response element and negatively interferes with PPAR gamma signalling in breast cancer cells[END_REF][START_REF] Bonofiglio | Peroxisome proliferator-activated receptor-gamma activates p53 gene promoter binding to the nuclear factor-kappaB sequence in human MCF7 breast cancer cells[END_REF]. In the last report, where we explored the involvement of the p53 pathway, it appears that the PPARc-mediated apoptosis is not completely blocked by using the p53 antisense, suggesting that final PPARc action in this concern is complex and it may require a multifactorial coordination of different signalling cascades. In this finding, we investigated the potential of PPARc in inducing apoptotic events through a direct involvement of the Fas/ FasL extrinsic apoptotic pathway in breast cancer cells.

The Fas/FasL signalling system plays an important role in chemotherapy-induced apoptosis in several different cell types [START_REF] Kasibhatla | DNA damaging agents induce expression of Fas ligand and subsequent apoptosis in T lymphocytes via the activation of NF-kappa B and AP-1[END_REF][START_REF] Mo | DNA damage signals induction of fas ligand in tumor cells[END_REF]. FasL is a member of the TNF superfamily that induces apoptosis in susceptible cells upon crosslinking of its own receptor, Fas [START_REF] Nagata | Apoptosis by death factor[END_REF][START_REF] Chinnaiyan | Portrait of an executioner: the molecular mechanism of FAS/APO-1-induced apoptosis[END_REF][START_REF] Suda | Molecular cloning and expression of the FAS Ligand, a novel member of the tumor necrosis factor family[END_REF][START_REF] Nagata | The fas death factor[END_REF]. FasL is expressed on the cell membrane surface of activated T lymphocytes and cancer cells [START_REF] Hahne | Melanoma cell expression of Fas (Apo-1/ CD95) ligand: implications for tumor immune escape[END_REF][START_REF] Oshimi | Fas ligand is expressed in normal breast epithelial cells and is frequently up-regulated in breast cancer[END_REF][42]. Significantly, constitutive down-regulation of Fas is involved in drug resistance [START_REF] Landowski | Selection for drug resistance results in resistance to Fas-mediated apoptosis[END_REF] and associated with a poor prognosis in breast cancer [START_REF] Toillon | Normal breast epithelial cells induce apoptosis of breast cancer cells via Fas signaling[END_REF]. It has been described that breast cancer cells may be Fas sensitive or Fas insensitive and divergent data are presented on FasL expression in these cells [START_REF] Keane | Fas expression and function in normal and malignant breast cell lines[END_REF][START_REF] Toillon | Normal breast epithelial cells induce apoptosis of breast cancer cells via Fas signaling[END_REF]. In our MCF7 cells, positive to Fas and FasL, we found that BRL through PPARc activation upregulated FasL at both protein and mRNA levels. Despite the FasL relevance, its cell and tissue distribution, and the apparent differences of its expression in a cell-specific manner, little knowledge is available how FasL expression is regulated. Recently, a downregulation of FasL by BRL has been reported in focal cerebral ischemia [START_REF] Chu | Peroxisome proliferator-activated receptor-gamma-agonist, rosiglitazone, promotes angiogenesis after focal cerebral ischemia[END_REF]. It was also showed that prostaglandin 15-deoxy-delta 12,14-prostaglandin J2, a natural PPARc ligand inhibits FasL gene expression in T lymphocytes via a PPARc-independent mechanisms [START_REF] Cippitelli | The cyclopentenone-type prostaglandin 15deoxy-delta 12,14-prostaglandin J2 inhibits CD95 ligand gene expression in T lymphocytes: interference with promoter activation via peroxisome proliferator-activated receptor-gammaindependent mechanisms[END_REF]. Indeed it is quite common for biological signalling pathways to vary depending on tissue type or cell condition.

We demonstrated that BRL increased the transactivation of FasL promoter in a dose-related and in a PPARc-dependent manner. Promoter deletion analysis of FasL has delineated a minimal promoter fragment spanning nucleotides from -318 to -237 bp, which was responsible for BRL activity. Indeed, the abovementioned region included three DNA motifs known to bind the transcription factors Sp1, NFAT and NFkB [START_REF] Mcclure | Constitutive Fas ligand gene transcription in Sertoli cells is regulated by Sp1[END_REF]. NFkB was reported to physically interact with PPARc [START_REF] Chung | Oxidized low density lipoprotein inhibits interleukin-12 production in lipopolysaccharide-activated mouse macrophages via direct interactions between peroxisome proliferator-activated receptorand nuclear factor-B[END_REF], which in some circumstances binds to DNA cooperatively with NFkB [START_REF] Bonofiglio | Peroxisome proliferator-activated receptor-gamma activates p53 gene promoter binding to the nuclear factor-kappaB sequence in human MCF7 breast cancer cells[END_REF][START_REF] Chung | Oxidized low density lipoprotein inhibits interleukin-12 production in lipopolysaccharide-activated mouse macrophages via direct interactions between peroxisome proliferator-activated receptorand nuclear factor-B[END_REF][START_REF] Coutureir | Interleukin 1ß induces type II-secreted phospholipase A 2 gene in vascular smooth muscle cells by a nuclear factor B and peroxisome proliferator-activated receptormediated process[END_REF]. NFAT and Sp1 are critical factors responsible for FasL gene activation [START_REF] Xiao | FasL promoter activation by IL-2 through SP1 and NFAT but not Egr-2 and Egr-3[END_REF]. NFAT is a family of related transcription factors that plays a central role in regulating the immune response and it has been demonstrated the involvement of NFAT in FasL transcription [START_REF] Mcclure | Constitutive Fas ligand gene transcription in Sertoli cells is regulated by Sp1[END_REF][START_REF] Rengarajan | Sequential involvement of NFAT and Egr transcription factors in FasL regulation[END_REF]. Several studies indicate that many mammalian gene types are controlled by Sp1, including genes for structural proteins, metabolic enzymes, cell cycle regulators, transcription factors, growth factors, surface receptors, and others [START_REF] Black | Sp1 and kruppel-like factor family of transcription factors in cell growth regulation and cancer[END_REF]. PPARc has been documented to functionally interact with Sp1 to modulate gene expression [START_REF] Sugawara | Transcription suppression of thromboxane receptor gene by peroxisome proliferator-activated receptor-c via an interaction with Sp1 in vascular smooth muscle cells[END_REF][START_REF] Sassa | Bifunctional properties of peroxisome proliferator-activated receptor c1 in KDR gene regulation mediated via interaction with both Sp1 and Sp3[END_REF]. These findings are in agreement with our results since the PPARc-mediated transactivation of FasL promoter was abolished by using mithramycin and a construct deleted of the Sp1 site. By EMSA studies with nuclear extract from MCF7 cells, a binding of PPARc to the Sp1 sequence located within the FasL promoter was observed, it was enhanced by BRL and immunodepleted by both PPARc and Sp1 Abs, addressing the coexistence of the two proteins in the DNA binding complexes. In addition, the interaction between PPARc and FasL promoter was supported by ChIP assay where BRL treatment also increases the binding of RNA-Pol II to this promoter gene addressing a positive transcriptional regulation mediated by PPARc.

Engagement of the FasL homotrimer to three Fas molecules induces apoptosis by clustering of the receptor's death domains [START_REF] Green | Apoptotic pathways: the roads to ruin[END_REF][START_REF] Nagata | Apoptosis by death factor[END_REF]. This leads to the binding of FADD and caspase 8 to the receptor [START_REF] Green | Apoptotic pathways: the roads to ruin[END_REF]. In the present study, we obtained the active cleavage of caspase 8 under BRL treatment, that was no longer detectable in the presence of GW as well as inhibiting both the expression of FasL and PPARc by the respective RNAis. Previous works from our group have identified important pathways underlying the role of PPARc in cell growth, cycle arrest and apoptosis in MCF7 breast cancer cells [START_REF] Bonofiglio | Estrogen receptor alpha binds to peroxisome proliferator-activated receptor (PPAR) response element and negatively interferes with PPAR gamma signalling in breast cancer cells[END_REF][START_REF] Bonofiglio | Peroxisome proliferator-activated receptor-gamma activates p53 gene promoter binding to the nuclear factor-kappaB sequence in human MCF7 breast cancer cells[END_REF]. First, we evidenced an opposite effects of estrogen receptor (ER) a and PPARc on the regulation of the PI3K/AKT pathway, classically involved in cell survival and proliferation, eliciting divergent responses in presence of the cognate ligands 17bestradiol and BRL respectively in estrogen-dependent MCF7 cells [START_REF] Bonofiglio | Estrogen receptor alpha binds to peroxisome proliferator-activated receptor (PPAR) response element and negatively interferes with PPAR gamma signalling in breast cancer cells[END_REF]. Subsequently, we showed that BRL promotes the growth arrest and apoptosis in MCF7 cells through a crosstalk between p53 and PPARc. Our results indicated that in a consecutive series of events BRL: (1) up-regulates the expression of p53 and (2) its effector p21WAF1/Cip1, (3) triggers the cleavage of caspase 9, an important component of the intrinsic apoptotic pathway [START_REF] Bonofiglio | Peroxisome proliferator-activated receptor-gamma activates p53 gene promoter binding to the nuclear factor-kappaB sequence in human MCF7 breast cancer cells[END_REF]. Finally, the DNA fragmentation assay clearly evidenced that PPARc was involved in the apoptotic process triggered by BRL since this effect was completely reversed by GW, while the apoptosis was only partially abolished inhibiting p53 expression by AS/p53, a plasmid encoding for p53 antisense [START_REF] Bonofiglio | Peroxisome proliferator-activated receptor-gamma activates p53 gene promoter binding to the nuclear factor-kappaB sequence in human MCF7 breast cancer cells[END_REF]. The reported finding evidenced that the cross talk between PPARc and p53 is responsible, at least in part, in the BRL-induced apoptosis in MCF7 cells [START_REF] Bonofiglio | Peroxisome proliferator-activated receptor-gamma activates p53 gene promoter binding to the nuclear factor-kappaB sequence in human MCF7 breast cancer cells[END_REF] and that alternative signalling cascades may be involved, like the Fas/FasL signalling that triggers the extrinsic apoptosis pathway, as we observed in this study. Interestingly, PPARc-induced apoptosis through FasL appears to be a general mechanism in breast cancer cells since it occurs in MCF7, MDA and BT20 cells.

In conclusion, we have shown that FasL expression is induced by PPARc through the binding to the transcription factor Sp1, demonstrating for the first time that PPARc triggers apoptotic events in breast cancer cells via Fas/FasL signalling pathway. However, the genomic response to PPARc activation remains complex and the understanding of the interactions between PPARc and other factors amplifies our knowledge on genes participating in cellcycle modulation and apoptosis. The data presented place FasL as a novel molecular target to add to the variety of anticancer activities mediated by PPARc and further candidate PPARc-ligands for the treatment of patients with ERa+ and ERa-breast cancer.

Fig. 1

 1 Fig. 1 BRL up-regulates FasL protein and mRNA expression in MCF7 cells. (a) Immunoblots of FasL and Fas from MCF7 breast cancer cells. (b) Immunoblots of FasL from MCF7 cells treated for 24 h with vehicle (-), increasing BRL concentrations, 10 lM GW alone or in combination with 1 lM BRL. b-actin was used as loading control. The side panel shows the quantitative representation of data (mean ± S.D.) of three independent experiments including that of b. (c) Semiquantitative RT-PCR evaluation of FasL mRNA expression. MCF7 cells were treated as in b. 36B4 mRNA levels were determined as control. The side panel shows the quantitative representation of data (mean ± S.D.) of three independent experiments including that of c after densitometry and correction for 36B4 expression. *P \ 0.05 BRL-treated vs untreated cells **P \ 0.01 BRL-treated vs. untreated cells

Fig. 2

 2 Fig. 2 BRL transactivates FasL promoter gene in MCF7 cells. (a) Schematic map of the FasL promoter fragments used in this study. (b) Basal transcriptional activity of the different FasLluciferase report constructs (Luc-0, Luc-3 and Luc-4). (c) MCF7 cells were treated for 24 h with vehicle (-), increasing BRL concentrations, 10 lM GW alone or in combination with 1 lM BRL. (d) MCF7 cells were treated for 24 h with vehicle (-), 1 lM BRL, 100 nM mithramycin (M) alone or in combination with 1 lM BRL. The luciferase activities were normalized to the Renilla luciferase as internal transfection control and data were reported as( RLU values. Columns are mean ± S.D. of three independent experiments performed in triplicate. *P \ 0.05 BRL-treated vs untreated cells, **P \ 0.01 BRL-treated vs untreated cells. RLU, Relative Light Units

Fig. 3

 3 Fig. 3 PPARc binds to Sp1 site within FasL promoter region in EMSA. Nuclear extracts from MCF7 cells (lane 1) were incubated with a labeled sequence containing bases -288 to -263 of the wildtype FasL promoter and subjected to electrophoresis in a 6% polyacrylamide gel. In lane 2, nuclear extracts from MCF7 were treated with 1 lM BRL. Competition experiments were performed adding as competitor a 100-fold molar excess of unlabeled FasL probe (lane 3) or 100 nM mithramycin (M) (lane 7). An excess of three unlabeled oligonucleotides DSp1 (lane 4), DNFAT (lane 5) and DNFkB (lane 6), was used. Anti-PPARc and anti-Sp1 Abs were incubated with nuclear extracts from MCF7 cells treated with 1 lM BRL (lanes 8 and 9, respectively). Lane 10 contains probe alone

Fig. 4

 4 Fig. 4 Functional interaction between PPARc and FasL promoter. (a) MCF7 cells were treated as indicated, then cross-linked with formaldehyde and lysed. The soluble chromatin was immunoprecipitated with the anti-PPARc Ab, (Re-ChIP with the anti-RNA Pol II and anti-Sp1 Abs). The FasL promoter sequence containing Sp1 was detected by PCR with specific primers, as described in Materials and Methods. To control input DNA, FasL promoter was amplified from 30 ll of initial preparations of soluble chromatin (before immunoprecipitations). In the bottom of the panel quantitative representation of data of three independent experiments (mean ± S.D.) after densitometry. *P \ 0.05 BRL-treated vs. untreated cells. M: mithramycin. N, negative control provided by PCR amplification without DNA sample. (b) Immunoblots of Sp1and PPARc from MCF7 cells treated with BRL 1 lM, in which chromatin was immunoprecipitated with the anti-PPARc antibody and IgG

Fig. 6

 6 Fig. 6 BRL induces FasL upregulation through PPARc in breast cancer cells. (a) FasL and Fas protein expression (evaluated by WB) in MDA and BT20 breast cancer cells. (b) MDA and BT20 cells were treated for 48 h as indicated, or transfected with a 25-nucleotide of RNA interference (RNAi) targeted human PPARc mRNA sequence or with control RNAi as reported in Materials and Methods. b-actin was used as loading control. One of three similar experiments is presented. (c) DNA laddering was performed in MDA and BT20 cells treated for 72-h as indicated, or transfected with PPARc or control RNAis