Gizeh Pérez-Tenorio

Elin Karlsson

Marie Ahnström Waltersson

Birgit Olsson

Birgitta Holmlund

Bo Nordenskjöld

Tommy Fornander

Lambert Skoog

Olle Stål
email: olle.stal@liu.se.

Clinical potential of the mTOR targets S6K1 and S6K2 in breast cancer

Keywords: mTOR, S6 kinase, 17q21-23, 11q13, gene amplification, tamoxifen response

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Introduction

The Mammalian Target of Rapamycin (mTOR) is a serine/threonine kinase, which in response to growth factors, hormones, nutrients, hypoxia and energy (ATP) regulates cell growth, proliferation and metabolism through translational control of essential proteins [START_REF] Ma | Molecular mechanisms of mTOR-mediated translational control[END_REF]. mTOR is a critical effector in several cellular functions commonly deregulated in cancer, and multiple alterations resulting in overstimulation of the pathway have been described [START_REF] Guertin | Defining the role of mTOR in cancer[END_REF]. Two major regulators of mTOR function, the RAS/MAPK and PI3K/AKT signaling pathways, are constitutively activated in many cancers. Mutations in the PIK3CA gene (encoding the p110 subunit of the PI3K), PTEN loss and aberrant activation or expression of AKT are some of these alterations found in breast cancer [START_REF] Perez-Tenorio | PIK3CA mutations and PTEN loss correlate with similar prognostic factors and are not mutually exclusive in breast cancer[END_REF][START_REF] Saal | PIK3CA mutations correlate with hormone receptors, node metastasis, and ERBB2, and are mutually exclusive with PTEN loss in human breast carcinoma[END_REF][START_REF] Samuels | High frequency of mutations of the PIK3CA gene in human cancers[END_REF]. Cross-talk between estrogen receptor (ER) signaling and the AKT/mTOR pathway is one suggested mechanism behind endocrine resistance in breast cancer [START_REF] Bärlund | Dual inhibition of mTOR and estrogen receptor signaling in vitro induces cell death in models of breast cancer[END_REF][START_REF] Kurokawa | ErbB (HER) receptors can abrogate antiestrogen action in human breast cancer by multiple signaling mechanisms[END_REF][START_REF] Schiff | Crosstalk between estrogen receptor and growth factor pathways as a molecular target for overcoming endocrine resistance[END_REF] and mTOR inhibition has been shown to increase the effect of endocrine treatments in both preclinical and clinical settings [START_REF] Chan | Phase II study of temsirolimus (CCI-779), a novel inhibitor of mTOR, in heavily pretreated patients with locally advanced or metastatic breast cancer[END_REF][START_REF] Degraffenried | Inhibition of mTOR activity restores tamoxifen response in breast cancer cells with aberrant Akt Activity[END_REF][START_REF] Baselga | Phase II randomized study of neoadjuvant everolimus plus letrozole compared with placebo plus letrozole in patients with estrogen receptor-positive breast cancer[END_REF]. Since multiple oncogenic cellular pathways converge on mTOR, an important prospect is a further dissection of the downstream signaling network of mTOR and to determine the clinical relevance of genetic alterations in the mTOR signaling pathway [START_REF] Guertin | Defining the role of mTOR in cancer[END_REF].

The ribosomal S6 kinases S6K1 and S6K2 are well-known mTOR substrates, involved in regulation of the translational machinery [START_REF] Ma | Molecular mechanisms of mTOR-mediated translational control[END_REF][START_REF] Park | Regulation of ribosomal S6 kinase 2 by mammalian target of rapamycin[END_REF][START_REF] Jastrzebski | Coordinate regulation of ribosome biogenesis and function by the ribosomal protein S6 kinase, a key mediator of mTOR function[END_REF][START_REF] Lee-Fruman | Characterization of S6K2, a novel kinase homologous to S6K1[END_REF]. S6K1 and S6K2 share 70 % overall amino acid identity, whereas the catalytic domains have even higher sequence homology with > 83 % overlapping residues. The domain structure and the several phosphorylation sites are also conserved and are found in the corresponding drosophila dS6K, indicating that the two S6K isoforms present in mammals result from gene duplication [START_REF] Jastrzebski | Coordinate regulation of ribosome biogenesis and function by the ribosomal protein S6 kinase, a key mediator of mTOR function[END_REF][START_REF] Shima | Disruption of the p70(s6k)/p85(s6k) gene reveals a small mouse phenotype and a new functional S6 kinase[END_REF]. Both kinases phosphorylate the 40S ribosomal protein S6 and are believed to have overlapping functions; however there are also data indicating divergence in their biological activities. In contrast to S6K1, S6K2 contains a proline-rich sequence, homologous to a sequence in the p85 subunit of PI3K, allowing interactions with SH3 domain-containing proteins [START_REF] Jastrzebski | Coordinate regulation of ribosome biogenesis and function by the ribosomal protein S6 kinase, a key mediator of mTOR function[END_REF]. Knock-out of S6K1 in mice, as well as drosophila dS6K has been connected to a reduction in animal body size during embryogenesis, as a result of a decrease in individual cell size [START_REF] Shima | Disruption of the p70(s6k)/p85(s6k) gene reveals a small mouse phenotype and a new functional S6 kinase[END_REF][START_REF] Montagne | Drosophila S6 kinase: a regulator of cell size[END_REF]. In contrast, S6K2 -/-mice had normal or slightly increased body size [START_REF] Pende | S6K1(-/-)/S6K2(-/-) mice exhibit perinatal lethality and rapamycin-sensitive 5'-terminal oligopyrimidine mRNA translation and reveal a mitogen-activated protein kinase-dependent S6 kinase pathway[END_REF]. Of note however, S6K1-deficient mice showed a significant upregulation of S6K2 protein in several tissues, suggesting a compensatory mechanism, which may explain why the phenotype of size reduction was mostly overcome by adulthood [START_REF] Shima | Disruption of the p70(s6k)/p85(s6k) gene reveals a small mouse phenotype and a new functional S6 kinase[END_REF]. Deletion of both S6K1 and S6K2 in mice, as well as dS6K in drosophila has been shown semilethal, severely reducing the viability. In contrast, no difference in lethality of S6K1 or S6K2 deficient mice have been seen, supporting the compensatory and essential roles for the kinases in normal development [START_REF] Montagne | Drosophila S6 kinase: a regulator of cell size[END_REF][START_REF] Pende | S6K1(-/-)/S6K2(-/-) mice exhibit perinatal lethality and rapamycin-sensitive 5'-terminal oligopyrimidine mRNA translation and reveal a mitogen-activated protein kinase-dependent S6 kinase pathway[END_REF].

The genes RPS6KB1 (S6K1) and RPS6KB2 (S6K2) are situated in the chromosomal regions 17q21-23 and 11q13, which are commonly amplified in several malignancies. In breast cancer, HER2 and CCND1 may be the most well-known oncogenes in these areas, where they are found amplified in 20-30% [START_REF] Lofts | c-erbB2 amplification and overexpression in human tumors[END_REF][START_REF] Singleton | Clinical and pathologic significance of the c-erbB-2 (HER-2/neu) oncogene[END_REF][START_REF] Slamon | Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene[END_REF] and 10-15% [START_REF] Al-Kuraya | Prognostic relevance of gene amplifications and coamplifications in breast cancer[END_REF][START_REF] Bostner | Amplification of CCND1 and PAK1 as predictors of recurrence and tamoxifen resistance in postmenopausal breast cancer[END_REF][START_REF] Jirström | Adverse effect of adjuvant tamoxifen in premenopausal breast cancer with cyclin D1 gene amplification[END_REF] of cases, respectively.

S6K1 amplification [START_REF] Bärlund | Detecting activation of ribosomal protein S6 kinase by complementary DNA and tissue microarray analysis[END_REF][START_REF] Van Der Hage | Overexpression of P70 S6 kinase protein is associated with increased risk of locoregional recurrence in nodenegative premenopausal early breast cancer patients[END_REF] and S6K1 protein overexpression [START_REF] Bärlund | Detecting activation of ribosomal protein S6 kinase by complementary DNA and tissue microarray analysis[END_REF][START_REF] Van Der Hage | Overexpression of P70 S6 kinase protein is associated with increased risk of locoregional recurrence in nodenegative premenopausal early breast cancer patients[END_REF][START_REF] Noh | Activation of the mTOR signaling pathway in breast cancer and its correlation with the clinicopathologic variables[END_REF] has earlier been associated with a worse outcome in breast cancer, but nothing has been reported about S6K2 in this context. Due to the location of S6K2 in a chromosomal region commonly amplified in malignancies, and the high homology between S6K1 and S6K2, one may hypothesize that also S6K2 could be of clinical importance. Consequently, the aim of this study was to evaluate the possible alterations of the mTOR targets S6K1 and S6K2 in postmenopausal breast cancer. S6K1 and S6K2 gene copy number was determined by fast real-time PCR in 207 stage II breast tumors; whereas S6K2 protein expression was detected by immunohistochemistry in 792 node-negative breast cancers. The prognostic and treatment predictive value regarding tamoxifen and radiotherapy was explored.

Materials and methods

In the following section the method procedures are briefly covered, and a detailed description can be found in Supplementary Methods. Study design and presentation of results are in line with the Reporting recommendations for tumor marker prognostic studies (REMARK) guidelines [START_REF] Mcshane | REporting recommendations for tumor MARKer prognostic studies (REMARK)[END_REF].

Patients

The patient materials used to study S6K1/S6K2 gene amplification and S6K2 protein expression were previously reported in detail [START_REF] Rutqvist | Long-term follow-up of the Stockholm randomized trials of postoperative radiation therapy versus adjuvant chemotherapy among 'high risk' pre-and postmenopausal breast cancer patients[END_REF][START_REF] Rutqvist | Long-term follow-up of the randomized Stockholm trial on adjuvant tamoxifen among postmenopausal patients with early stage breast cancer[END_REF]. Briefly, accrual of high-risk and lowrisk postmenopausal patients started in November 1976 and ended in April 1990. The lowrisk group included patients without positive lymph nodes and a tumor diameter ≤ 30 mm, while the high-risk group consisted of patients with either histological verified lymph node metastases or a tumor diameter > 30 mm. Both patient cohorts were randomized to receive adjuvant tamoxifen or no endocrine treatment. Furthermore, the high-risk group was randomized to cyclophosphamide-methotrexate-5-fluorouracil (CMF) chemotherapy or radiotherapy (RT) (Fig. 1).

The S6K1/S6K2 gene copy number analysis comprised a subset of patients from the high-risk group, from whom frozen tumor tissue was still available after hormone receptor assays and other biochemical analyses. Furthermore, all samples included were judged to contain > 50% of malignant cells (n=207). From these, 34 tumors with 11q13 amplification were selected, out of which 23 were available for S6K2 mRNA expression analysis. Formalinfixed and paraffin-embedded tumors from the low-risk group (n=912) were used for S6K2 protein expression analysis (Fig. 1).

The two subsets showed no major differences in comparison with all the postmenopausal patients in the trial in terms of tumor characteristics and treatment. Median follow-up times were 18 years for the low-risk patients and 11 years for the high-risk patients.

This study was approved by the local ethical committee at the Karolinska Institute.

DNA and mRNA preparation

Extraction of genomic DNA was performed as described before [START_REF] Askmalm | Mutation and accumulation of p53 related to results of adjuvant therapy of postmenopausal breast cancer patients[END_REF] and DNA concentration was estimated using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies). For mRNA preparation, fresh frozen tumor tissue was homogenized with a microdismembrator (B Braun) and total RNA was isolated using the mirVanaTM miRNA Isolation kit (Ambion), according to manufacturer's recommendations. Purified RNA was eluted in nuclease-free water and RNasin® Ribonuclease Inhibitor (Promega) was added before storage in -70 C. RNA quantity and quality was assessed with an Agilent 2100 Bioanalyzer (Agilent biosystems).

Evaluation of S6K1/S6K2 gene copy number

S6K1 and S6K2 gene copy number was determined in 206 respectively 207 available breast tumors, using quantitative real-time PCR. Details of the performance can be found in Supplementary Methods.

S6K2 mRNA quantification

S6K2 mRNA levels were measured in 23/34 available samples selected for amplification in the 11q13 area, using quantitative real-time PCR. Reverse transcription and mRNA quantitation is further described in Supplementary Methods.

Immunohistochemistry and immunoblotting

Formalin-fixed and paraffin-embedded tumors from the low-risk group (n=912) were used for S6K2 protein expression analysis. Procedures for immunochemical staining of S6K2 as well as Cyclin D1, and evaluation of antibody specificity using immunoblotting are presented in detail in Supplementary Methods. Preparation of breast cancer tissue microarrays (TMA) and evaluation of ER, progesterone receptor (PgR) and HER2 protein expression have been described previously [START_REF] Jansson | Ratio of 17HSD1 to 17HSD2 protein expression predicts the outcome of tamoxifen treatment in postmenopausal breast cancer patients[END_REF].

Statistical analysis

Spearman's rank order correlation was used to determine the association between S6K2 gene copy number and mRNA expression levels. The relationships between different grouped variables were assessed by the Chi-square test or Chi-square test for trend, when appropriate. The product-limit method was used for estimation of cumulative probabilities of recurrence-free survival (RFS) and distant recurrence-free survival (DRFS). Differences in survival between groups were tested with the log-rank test. Univariate and multivariate analysis of event rates was performed with Cox proportional hazard regression. This was also applied for interaction analysis of different factors and treatment by including the variables X (potential predictive factor), treatment, and the interaction variable (X * treatment). All the procedures were comprised in STATISTICA, version 8.0, StatSoft, Inc. (2007). The criterion for statistical significance was P<0.05.

Results

S6K1 and S6K2 gene amplification

S6K1 and S6K2 gene amplification was analyzed in 206 and 207 high-risk breast tumors, respectively. Amplification (≥ 4 copies) of the S6K1 gene could be detected in 22/206 cases (10.7%) while the S6K2 gene was amplified in 9/207 cases (4.3%). Gain (≥ 3 copies) was observed in 44 cases for both S6K1 (21.4%) and S6K2 (21.3%). S6K1 amplification varied from 4 to 21 estimated copies of the gene, while S6K2 amplification was in the range from 4 to 9 copies. Amplification of S6K1 and S6K2 were mutually exclusive events in the cohort (Table 1), why S6K1 or S6K2 amplification was detected in 31/206 cases (15%). S6K1 gain and/or S6K2 gain occurred in 74/205 cases (36%).

S6K1 amplification (Table 1) was significantly associated with HER2 gene amplification (P=0.025) and HER2 protein expression (P=0.014) and tended to be inversely correlated to CCND1 amplification (P=0.065). Also S6K1 gain correlated significantly to HER2 amplification (P=0.007) and was borderline associated with high S-phase fraction (P=0.062) and large tumor size (P=0.067). S6K2 gene copy number was significantly associated with S6K2 mRNA expression levels (P=0.0001). Amplification of S6K2 (Table 1) correlated to positive ER status (P=0.046) whereas both S6K2 amplification and S6K2 gain correlated to CCND1 amplification (p<0.00001 and P=0.00003). S6K2 gain was also significantly associated with a high S-phase fraction (P=0.027). The combination variable S6K1 or S6K2 amplification, as well as S6K1 and/or S6K2 gain was inversely correlated to PIK3CA mutations (P=0.012 and P=0.029), whereas the latter combination variable also correlated significantly to high S-phase fraction (P=0.016).

S6K2 protein expression

S6K2 protein expression was analyzed with immunohistochemistry in 792/912 lowrisk breast tumors. Nuclear and cytoplasmic S6K2 were detected in 38% and 76% of the tumors respectively (Fig. 2 a-c). The S6K2 antibody was evaluated by immunoblotting in order to disregard the presence of unspecific bands or cross-reaction with the S6K1 protein (Fig. 2 d). Nuclear S6K2 was positively correlated with ER+ (P< 0.00001), PgR+ (P< 0.00001) status and nuclear Cyclin D1 protein expression (P<0.00001), whereas it was inversely correlated with HER2 protein expression (P=0.013). Cytoplasmic S6K2 correlated with ER+ status (P=0.009) and nuclear Cyclin D1 protein expression (P<0.00001).

Survival analysis

In an univariate analysis including all high-risk patients, S6K1 gene amplification tended to confer a higher risk of developing distant metastasis (HR=1.63, 95% CI, 0.92-2.85, P=0.092, Fig. 3 a) whereas S6K1 gain was significantly associated with increased risk of distant recurrence (HR=1.62, 95% CI, 1.05-2.52, P=0.031, Fig. 3 b). Amplification of S6K2 significantly predicted a higher risk of distant recurrence in breast cancer (HR=2.70, 95% CI, 1.24-5.83, P=0.012, Fig. 3 c), whereas this could not be seen for S6K2 gain (HR=1.29, 95% CI, 0.83-2.01, P=0.26, Fig. 3 d).

The combination variable S6K1 or S6K2 amplification was significantly associated with poor DRFS (HR=1.98, 95% CI, 1.22-3.20, P=0.006, Fig. 3 e) and this was also true for the combination variable S6K1 and/or S6K2 gain (HR=1.61, 95% CI, 1.09-2.37, P=0.016, Fig. 3 f). Among patients with ER positive tumors, the combination variables tended to have an even stronger prognostic value in terms of DRFS (S6K1 or S6K2 amplification: HR=2.23, 95% CI, 1.29-3.88, P=0.0044; S6K1 and/or S6K2 gain: HR=1.90, 95% CI, 1.18-3.05, P=0.008).

In a multivariate analysis, including HER2 and CCND1 amplification as well as treatment, among other common variables, S6K2 amplification remained an independent prognostic factor of increased risk for distant recurrence, whereas S6K1 gene amplification reached borderline significance (Table 2).

The combination variables S6K1 or S6K2 amplification (HR=2.11, 95% CI, 1.27-3.50, P=0.004) as well as S6K1 and/or S6K2 gain (HR=1.54, 95% CI, 1.00-2.38, P=0.049) also resulted as independent prognostic factors in an analogous multivariate analysis.

In the cohort of low-risk patients, S6K2 protein expression did not show any prognostic value (data not shown).

Treatment prediction

As a result of the low number of cases with S6K2 amplification, S6K2 gain was considered in analyses of treatment prediction. The benefit from tamoxifen was evident for high-risk patients having ER positive tumors with S6K2 gain regarding DRFS, whereas no significant tamoxifen response could be seen in the S6K2 negative group (Fig. 4 a,b). In the low-risk group, nuclear S6K2 protein expression was associated with an increased benefit from tamoxifen among patients with ER+/PgR+ tumors (Fig. 4 c,d). However in the ER+/PgR-group, nuclear S6K2 expression was rather an indicator of decreased tamoxifen responsiveness (Fig. 4 e,f). In an interaction test, S6K2 gain had borderline significance as a predictor of increased tamoxifen efficacy, using DRFS as the end-point (Table 3) and the interaction reached significance in terms of RFS (P=0.026, data not shown). Also nuclear S6K2 protein expression interacted significantly with the benefit from tamoxifen among the low-risk ER+/PgR+ patients, whereas a trend for a negative interaction between nuclear S6K2

and tamoxifen efficacy could be seen in the ER+/PgR-group (Table 4). S6K1 gene amplification alone did not show any predictive value regarding tamoxifen treatment (Table 3), however a trend was seen for the combination variable S6K1 amplification and/or S6K2 gain to predict increased benefit from tamoxifen (Table 3), and the test for interaction reached significance using RFS as the primary end-point (P=0.046, data not shown).

In terms of loco-regional control, the patients with normal S6K1 gene copy number responded significantly better to radiotherapy compared to chemotherapy in contrast to the patients harboring tumors with S6K1 amplification (Supplementary Table 1). Genomic amplification on 17q21-23 including S6K1 and/or HER2 gene amplification, also indicated poor response to radiotherapy (Supplementary Fig. 1). Both 17q21-23 and S6K1 amplification interacted significantly with the benefit from radiotherapy (Supplementary Table 1). A similar trend was seen for S6K2, where a normal copy number was associated with a significant benefit from radiotherapy compared to chemotherapy, whereas S6K2 gain was not (Supplementary Table 1). Though, no significant interaction between S6K2 or the combination variable S6K1 amplification and/or S6K2 gain, and radiotherapy, was evident (Supplementary Table 1).

Discussion

Genomic amplifications occur frequently and non-randomly in tumors and are expected to be essential for the development and progression of malignancy. In breast cancer, 17q21-23 and 11q13 are commonly amplified chromosomal regions where HER2 and CCND1 may be the most well-known oncogenes [START_REF] Lofts | c-erbB2 amplification and overexpression in human tumors[END_REF][START_REF] Singleton | Clinical and pathologic significance of the c-erbB-2 (HER-2/neu) oncogene[END_REF][START_REF] Slamon | Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene[END_REF][START_REF] Al-Kuraya | Prognostic relevance of gene amplifications and coamplifications in breast cancer[END_REF][START_REF] Bostner | Amplification of CCND1 and PAK1 as predictors of recurrence and tamoxifen resistance in postmenopausal breast cancer[END_REF][START_REF] Jirström | Adverse effect of adjuvant tamoxifen in premenopausal breast cancer with cyclin D1 gene amplification[END_REF]. The present study suggests a role for S6K1

and S6K2 as clinically valuable in these amplicons.

This is the first study to report amplification/gain of S6K2 and its correlation to an increased S6K2 mRNA expression in primary breast tumors. Amplification of the homologous S6K1 was detected in about 10 % of the tumors, which is in agreement with earlier studies where amplification of S6K1 also has been correlated to increased expression of the corresponding protein [START_REF] Bärlund | Detecting activation of ribosomal protein S6 kinase by complementary DNA and tissue microarray analysis[END_REF][START_REF] Van Der Hage | Overexpression of P70 S6 kinase protein is associated with increased risk of locoregional recurrence in nodenegative premenopausal early breast cancer patients[END_REF][START_REF] Sinclair | The 17q23 amplicon and breast cancer[END_REF]. S6K1 and S6K2 amplification were mutually exclusive events in the cohort, suggesting compensatory roles as tumor driving oncogenes.

The joint value of S6K1 and/or S6K2 gene copy number alterations was explored and appeared to be of clinical relevance.

Amplification and gain of S6K1 was significantly associated with HER2 gene amplification and HER2 protein overexpression. The possibility of S6K1 and HER2 coamplification has been discussed before due to their physical proximity [START_REF] Bärlund | Detecting activation of ribosomal protein S6 kinase by complementary DNA and tissue microarray analysis[END_REF]. S6K1 was identified as the first candidate oncogene in the 17q23 region [START_REF] Couch | Localization of PS6K to chromosomal region 17q23 and determination of its amplification in breast cancer[END_REF] and S6K1 or S6K1/HER2 amplification have been associated to a poor outcome in breast cancer [START_REF] Bärlund | Detecting activation of ribosomal protein S6 kinase by complementary DNA and tissue microarray analysis[END_REF].

S6K2 amplification and gain were strongly correlated to CCND1 amplification. The physical proximity of these two genes (2.2 Mb) suggests that they belong to the same amplicon or to frequently coamplified cores within the 11q13 area [START_REF] Albertson | Gene amplification in cancer[END_REF]. 11q13 amplification has in several studies been connected to positive ER status in breast cancer [START_REF] Elsheikh | CCND1 amplification and cyclin D1 expression in breast cancer and their relation with proteomic subgroups and patient outcome[END_REF][START_REF] Letessier | Frequency, prognostic impact, and subtype association of 8p12, 8q24, 11q13, 12p13, 17q12, and 20q13 amplifications in breast cancers[END_REF], which could also be confirmed here for amplification of S6K2. However, the mechanisms behind a possible interaction between 11q13 and ER remain to be elucidated.

Of note, amplification or gain of S6K1 or S6K2 were inversely correlated with the presence of PIK3CA mutations, indicating that deregulation of the S6 kinases may be an alternative and compensatory mechanism for PI3K/AKT stimulation in breast tumors.

S6K1 and S6K2 share structural homology although they exhibit differences in the C and N terminal domains [START_REF] Lee-Fruman | Characterization of S6K2, a novel kinase homologous to S6K1[END_REF]. The S6 kinases have earlier been observed in both cytoplasmic and nuclear compartments of malignant cells [START_REF] Lyzogubov | Immunohistochemical analysis of Ki-67, PCNA and S6K1/2 expression in human breast cancer[END_REF] where different S6K1 and 2 isoforms have been reported. S6K1 exists as p70 and p85 isoforms. Likewise, the two S6K2 isoforms p60/ I and p54/ II have been found in the cytoplasm and the nucleus [START_REF] Phin | Mutational analysis of ribosomal S6 kinase 2 shows differential regulation of its kinase activity from that of ribosomal S6 kinase 1[END_REF]. Since the known function of these proteins is so far coupled to phosphorylation of a ribosomal protein present in the cytoplasm, the role of the nuclear S6K1/2 is intriguing, suggesting the possibility of other substrates. S6K1 protein expression have earlier been correlated to S6K1 gene amplification [START_REF] Bärlund | Detecting activation of ribosomal protein S6 kinase by complementary DNA and tissue microarray analysis[END_REF][START_REF] Van Der Hage | Overexpression of P70 S6 kinase protein is associated with increased risk of locoregional recurrence in nodenegative premenopausal early breast cancer patients[END_REF] and associated with a worse outcome in breast cancer [START_REF] Bärlund | Detecting activation of ribosomal protein S6 kinase by complementary DNA and tissue microarray analysis[END_REF][START_REF] Van Der Hage | Overexpression of P70 S6 kinase protein is associated with increased risk of locoregional recurrence in nodenegative premenopausal early breast cancer patients[END_REF][START_REF] Noh | Activation of the mTOR signaling pathway in breast cancer and its correlation with the clinicopathologic variables[END_REF] but very little is known about S6K2 protein expression. In the present study, S6K2 protein could be detected in the nuclear and cytoplasmic compartments of breast tumor cells. In accordance with S6K2 amplification, S6K2 protein expression also correlated to Cyclin D1 expression and ER positive status, but also to PgR expression, implying a functional connection at the cellular level between S6K2 and ER signaling.

The current data confirm a role for both S6K1 and S6K2 amplification/gain as prognostic factors in breast cancer, possibly of greatest significance in the ER-positive subgroup. S6K2 amplification remained as an independent prognostic factor and S6K1 reached borderline significance in a multivariate analysis including HER2 and CCND1 amplification as well as treatment, among other common variables, demonstrating the individual contribution of the S6 kinases as potential oncogenes in the amplicons. The combination variables S6K1 or S6K2 amplification, as well as S6K1 and/or S6K2 gain, also remained independent prognostic factors in analogues analysis.

Anti-estrogen treatments are corner stones in the management of ER positive breast cancer, however de novo and acquired endocrine resistance remains a substantial problem.

Identifying new biomarkers for prediction of responsiveness to endocrine treatments is therefore of great importance [START_REF] Massarweh | Unraveling the mechanisms of endocrine resistance in breast cancer: new therapeutic opportunities[END_REF]. Results from this study indicate that the S6 kinases, in particular S6K2, may be relevant in this context. Increased S6K2 gene copy number and nuclear S6K2 expression was shown related to a better response to tamoxifen among patients with ER positive tumors. Interestingly, the ability of S6K2 to predict benefit from tamoxifen was restricted to the ER+/PgR+ subgroup among the low-risk cohort in the present study.

Among patients with ER+/PgR-tumors, nuclear S6K2 expression was rather connected to a worse response to endocrine treatment. This allows for the speculation that ER signaling in this subgroup may be driven in a hormone-independent manner, via cross-talk to intracellular signaling pathways including mTOR/S6K. mTOR inhibitors have been shown effective in combination with endocrine therapies in both clinical and preclinical studies [START_REF] Chan | Phase II study of temsirolimus (CCI-779), a novel inhibitor of mTOR, in heavily pretreated patients with locally advanced or metastatic breast cancer[END_REF][START_REF] Degraffenried | Inhibition of mTOR activity restores tamoxifen response in breast cancer cells with aberrant Akt Activity[END_REF][START_REF] Baselga | Phase II randomized study of neoadjuvant everolimus plus letrozole compared with placebo plus letrozole in patients with estrogen receptor-positive breast cancer[END_REF]. In the light of the present findings, S6K2 may have a role in predicting when this combination therapy is useful. S6K1 has earlier been implicated in the regulation of ER signaling by phosphorylating ERα-Ser 167, leading to increased ER transcriptional activity and cell growth in vitro [START_REF] Yamnik | S6 kinase 1 regulates estrogen receptor alpha in control of breast cancer cell proliferation[END_REF]. In addition, phosphorylation of ERα-Ser 167 has been associated with better response to tamoxifen [START_REF] Jiang | Phosphorylation of estrogen receptor-alpha at Ser167 is indicative of longer diseasefree and overall survival in breast cancer patients[END_REF][START_REF] Yamashita | Phosphorylation of estrogen receptor alpha serine 167 is predictive of response to endocrine therapy and increases postrelapse survival in metastatic breast cancer[END_REF], and a similar role for S6K2 in ER phosphorylation may be conceivable. The proline-rich motif found in S6K2 may support this speculation, since a proline rich, SH3 binding domain in certain ER coactivators have been shown essential for their function and interactions with ERα [START_REF] Zhou | The molecular basis of the interaction between the proline-rich SH3-binding motif of PNRC and estrogen receptor alpha[END_REF].

The HER2/PI3K/AKT signaling pathway has earlier been implicated in resistance to radiation-induced apoptosis in breast tumors [START_REF] Söderlund | Activation of the phosphatidylinositol 3kinase/Akt pathway prevents radiation-induced apoptosis in breast cancer cells[END_REF], and this can be reversed by the HER2

inhibitor trastuzumab [START_REF] Liang | Sensitization of breast cancer cells to radiation by trastuzumab[END_REF]. Results from the present study reveal that S6K1 may also be of interest in this context, in particular in connection to HER2 coamplification. A similar role for S6K2 cannot be excluded, however, the impact on radiosensitivity appear to be mainly connected to the 17q21-23 amplicon. Of note, the RAD51C gene is located about 1 Mb from S6K1, and the RAD51 DNA repair family has in both in vivo and in vitro studies been related to a poor sensitivity of radiation-induced apoptosis [START_REF] Söderlund | The BRCA1/BRCA2/Rad51 complex is a prognostic and predictive factor in early breast cancer[END_REF][START_REF] Russell | Gleevec-mediated inhibition of Rad51 expression and enhancement of tumor cell radiosensitivity[END_REF][START_REF] Taki | Antisense inhibition of the RAD51 enhances radiosensitivity[END_REF][START_REF] Ohnishi | In vitro and in vivo potentiation of radiosensitivity of malignant gliomas by antisense inhibition of the RAD51 gene[END_REF].

In conclusion, this study shows for the first time that S6K2 is amplified and overexpressed in breast tumors, which like S6K1 amplification may have prognostic significance. Resulting data demonstrate a role for the S6 kinases in predicting response of tamoxifen as well as radiotherapy treatment, but further studies are needed to uncover underlying mechanisms. The mTOR targets S6K1 and S6K2 may possess both compensatory and non-redundant functions associated with malignancy and therefore have potential as new prognostic and predictive markers in breast cancer.

Figure 1 Figure 2 Figure 3 Figure 4 Figure

 1234 Figure 1

Table 1 .

 1 S6K1 and S6K2 gene amplification (≥ 4 copies) in relation to clinicopathological

	factors and the PI3K/AKT pathway
		S6K1 amplification n (%)	S6K2 amplification n (%)
		-	+	Test for	-	+	Test for
				significance			significance
	Nodes
	-	18 (81.8) 4 (18.2) P=0.23	20 (90.9) 2 (9.1) P=0.25
	+	166 (90.2) 18 (9.8)		178 (96.2) 7 (3.8)
	Tumor Size
	20 mm	73 (90.1) 8 (9.9)	P=0.76	80 (96.4) 3 (3.6) P=0.67
	> 20 mm	111 (88.8) 14 (11.2)		118 (95.2) 6 (4.8)
	ER
	-	55 (90.2) 6 (9.8)	P=0.78	61 (100)	0 (0)	P=0.046
	+	127 (88.8) 16 (11.2)		135 (93.8) 9 (6.2)
	S-phase fraction a
	< 10%	96 (90.6) 10 (9.4) P=0.26	103 (97.2) 3 (2.8) P=0.26
	≥ 10%	69 (85.2) 12 (14.8)		76 (93.8) 5 (6.2)
	PIK3CA mutation b
	-	134 (87.1) 20 (12.9) P=0.081	146 (94.2) 9 (5.8) P=0.084
	+	47 (95.9) 2 (4.1)		49 (100)	0 (0)
	pAKT(Ser 473) c
	-(0%)	86 (88.7) 11 (11.3) P=0.82	92 (94.9) 5 (5.1) P=0.87
	+ (1-10%)	46 (93.9) 3 (6.1)		47 (95.9) 2 (4.1)
	++ (>10%)	50 (86.2) 8 (13.8)		57 (96.6) 2 (3.4)
	HER2 amplification d
	-	141 (91.6) 13 (8.4) P=0.025	147 (94.8) 8 (5.2) P=0.42
	+	35 (79.5) 9 (20.5)		43 (97.7) 1 (2.3)
	HER2 protein a
	-	134 (92.4) 11 (7.6) P=0.014	138 (94.5) 8 (5.5) P=0.25
	+	45 (80.4) 11 (19.6)		55 (98.2) 1 (1.8)
	CCND1 amplification e
	-	145 (87.4) 21 (12.6) P=0.065	165 (98.8) 2 (1.2) P< 0.00001
	+	24 (100)	0 (0)		17 (70.8) 7 (29.2)
	S6K2 amplification
	-	175 (89.3) 21 (10.7) P=0.30
	+	9 (100)	0 (0)
	a [50], b [3], c [51], d [52], e [22]

Table 2 .

 2 Multivariate analysis of distant recurrence using Cox proportional hazard

	regression
		HR (95% CI)	Test for significance
	Lymph node status N+ vs. N-	2.59 (1.09-6.15)	P=0.032
	Tumor size > 20 mm vs. ≤ 20 mm	1.77 (1.13-2.75)	P=0.012
	ER status ER+ vs. ER-	0.82 (0.51-1.29)	P=0.39
	HER2 gene amplification Amplified vs. nonamplified	1.63 (1.01-2.64)	P=0.045
	CCND1 gene amplification Amplified vs. nonamplified	0.95 (0.46-1.99)	P=0.90
	Tamoxifen vs. no Tamoxifen	0.77 (0.51-1.17)	P=0.22
	Chemotherapy vs. Radiotherapy	1.16 (0.76-1.76)	P=0.49
	S6K1 gene amplification Amplified vs. nonamplified	1.78 (0.98-3.22)	P=0.059
	S6K2 gene amplification Amplified vs. nonamplified	3.65 (1.40-9.54)	P=0.008

Table 3 .

 3 Cox proportional hazard regression of distant recurrence rate for patients with stage II, ER+ tumors, treated or not with adjuvant tamoxifen, in relation to S6K1 amplification ( 4 gene copies), S6K2 gain ( 3 gene copies) and the combination variable

	S6K1 amplification and/or S6K2 gain
		No. of patients Tamoxifen vs. no tamoxifen	Test for
			HR (95% CI)		interaction
	S6K1 amplification
	-	125	0.66 (0.40-1.10)	P=0.11
	+	16	0.62 (0.16-2.40)	P=0.49	P=0.94
	S6K2 gain
	-	110	0.80 (0.45-1.41)	P=0.44
	+	32	0.21 (0.08-0.53)	P=0.001	P=0.065
	S6K1 amplification
	and/or S6K2 gain
	-	96	0.81 (0.43-1.52)	P=0.52
	+	46	0.34 (0.26-0.74)	P=0.006	P=0.16

Table 4 .

 4 Cox proportional hazard regression of distant recurrence rate for patients with node-negative breast cancers, and ER+, ER+/PgR+ or ER+/PgR-tumors, respectively, treated or not with adjuvant tamoxifen, in relation to nuclear S6K2 protein expression

		No. of patients Tamoxifen vs. no tamoxifen	Test for
			HR (95% CI)	interaction
	ER+
	S6K2n-	337	0.54 (0.33-0.88) P=0.013
	S6K2n+	265	0.44 (0.21-0.78) P=0.007	P=0.52
	ER+/PgR+
	S6K2n-	165	0.60 (0.29-1.22) P=0.16
	S6K2n+	163	0.17 (0.07-0.42) P=0.0001	P=0.034
	ER+/PgR-
	S6K2n-	142	0.49 (0.24-1.00) P=0.049
	S6K2n+	80	1.33 (0.43-4.06) P=0.62	P=0.13

Acknowledgments

This study was supported by grants from the Swedish Cancer Foundation, Swedish Research Council and King Gustaf V Jubilee Fund.

Disclosure/conflict of interest

The authors have no conflicts of interest do disclose.


Antoinette Hollestelle
email: a.hollestelle@erasmusmc.nl

Cory Pelletier

Maartje Hooning

Ellen Crepin

Mieke Schutte

Maxime Look

J Margriet Collee

Anja Nieuwlaat

Lambert C J Dorssers

Caroline Seynaeve

Yurii S Aulchenko

John W M Martens

Ans M W Van Den Ouweland

Joanne B Weidhaas
email: joanne.weidhaas@yale.edu

Prevalence of the variant allele rs61764370 T>G in the 3'UTR of among Dutch , and non-/ breast cancer families

Keywords: KRAS-variant, let-7, breast cancer susceptibility, association, BRCA1

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

Introduction

Breast cancer is the most frequently diagnosed type of cancer in women from western countries and an appreciable fraction is attributable to genetic predisposition to the disease. Disease-causing mutations in the high-risk breast cancer susceptibility genes BRCA1 and BRCA2 together explain about 20% of the familial clustering of breast cancer. Population-based studies have estimated the cumulative risk of breast cancer to be 65% and 45% for BRCA1 and BRCA2 mutation carriers, respectively [START_REF] Antoniou | Average risks of breast and ovarian cancer associated with BRCA1 or BRCA2 mutations detected in case Series unselected for family history: a combined analysis of 22 studies[END_REF]. The penetrance of BRCA1 and BRCA2 mutations, however, is generally higher in women from families with multiple affected individuals, suggesting the presence of other risk-modifying factors in these families [START_REF] Ford | Risks of cancer in BRCA1mutation carriers. Breast Cancer Linkage Consortium[END_REF][START_REF] Ford | Genetic heterogeneity and penetrance analysis of the BRCA1 and BRCA2 genes in breast cancer families. The Breast Cancer Linkage Consortium[END_REF][START_REF] Struewing | The risk of cancer associated with specific mutations of BRCA1 and BRCA2 among Ashkenazi Jews[END_REF]. Indeed, it was shown that low-risk breast cancer susceptibility alleles exist that may act multiplicatively on the breast cancer risk for BRCA1 and BRCA2 mutation carriers [START_REF] Antoniou | Consortium of Investigators of Modifiers of B (2007) RAD51 135G-->C modifies breast cancer risk among BRCA2 mutation carriers: results from a combined analysis of 19 studies[END_REF][START_REF] Antoniou | Common breast cancer-predisposition alleles are associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers[END_REF].

Besides BRCA1 and BRCA2, three other high-risk breast cancer susceptibility genes have been recognized: p53, PTEN and STK11. However, these account for only a small fraction (less than 1%) of the familial breast cancer risk and are rarely observed outside the context of the cancer syndromes they cause (e.g. Li-Fraumeni syndrome, Cowden syndrome and Peutz-Jeghers syndrome, respectively). Another 5% of the familial breast cancer risk is explained by mutations in moderate-risk genes ATM, BRIP1, CHEK2, NBS1 and PALB2 and low-risk alleles such as those in or near the FGFR2, TNRC9/TOX3 and MAP3K1 genes [START_REF] Stratton | The emerging landscape of breast cancer susceptibility[END_REF][START_REF] Hollestelle | Discovering moderate-risk breast cancer susceptibility genes[END_REF]. This means that so far three quarters of the familial clustering of breast cancer remains unexplained. Importantly, twin studies suggested that a significant proportion of the remaining familial breast cancer risk is most probably due to genetic factors [START_REF] Ahlbom | Cancer in twins: genetic and nongenetic familial risk factors[END_REF][START_REF] Mack | Heritable breast cancer in twins[END_REF].

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that negatively regulate gene expression by binding partially complementary sites in the 3' untranslated region (UTR) of their target mRNAs. The importance of miRNAs in cancer is stressed by their widespread deregulation in virtually all cancer types [START_REF] Esquela-Kerscher | Oncomirs -microRNAs with a role in cancer[END_REF][START_REF] Trang | MicroRNAs as potential cancer therapeutics[END_REF]. Recently, a variant allele in the 3'UTR of the KRAS gene (rs61764370 T>G) was shown to disrupt a let-7 miRNA binding site leading to increased expression of KRAS in vitro and lower let-7 miRNA levels in vivo. [START_REF] Chin | A SNP in a let-7 microRNA complementary site in the KRAS 3' untranslated region increases non-small cell lung cancer risk[END_REF]. Consistent with the oncogenic nature of KRAS, the variant allele was shown to confer an increased risk for developing non-small cell lung cancer (NSCLC), as well as ovarian cancer ( [START_REF] Chin | A SNP in a let-7 microRNA complementary site in the KRAS 3' untranslated region increases non-small cell lung cancer risk[END_REF] and Ratner et al., in press), although the results in NSCLC were not replicated [START_REF] Nelson | KRAS mutation, KRAS-LCS6 polymorphism, and non-small cell lung cancer[END_REF]. In head and neck cancers, the KRAS-variant was shown to be a genetic marker of poor outcome as well [START_REF] Christensen | A let-7 microRNA-binding site polymorphism in the KRAS 3' UTR is associated with reduced survival in oral cancers[END_REF]. Because the frequency of the KRAS-variant was found to be increased among unselected ovarian cancer cases as well as within the context of hereditary breast and ovarian cancer (HBOC) families, we wondered whether the variant allele frequency might also be increased in breast cancer families. Therefore, we genotyped the KRAS-variant in breast cancer index cases from 268 BRCA1 families, 89 BRCA2 families and 685 non-BRCA1/BRCA2 families and compared its prevalence with 797 geographically-matched controls. We also investigated the association of the KRAS-variant with specific characteristics of BRCA1 families.

Materials and Methods

-4 -

Study population

Families with clustering of breast cancer were selected from the database of the Rotterdam Family Cancer Clinic at Erasmus University Medical Center -Daniel den Hoed Cancer Center in Rotterdam, representing the Southwestern part of the Netherlands. Selected families included at least two cases of female breast cancer or at least one case of female breast cancer and one case of ovarian cancer in first-or second-degree relatives. For each family, the youngest breast cancer patient who had been tested for BRCA1 and BRCA2 was assigned to be the index case. The control population was geographically matched and included 797 females from cystic fibrosis families who were either spouses of individuals at risk of being carrier of a CFTR mutation or individuals who were tested negative for a CFTR mutation. This unmatched case-control study was approved by the local ethical committee and all individuals gave full informed consent to search for susceptibility alleles.

Genotyping

DNA isolated from peripheral blood was amplified using a custom-made Taqman genotyping assay (Applied Biosystems, Foster City, CA) designed specifically to identify the T or variant G allele of the KRAS-variant (rs61764370). Forward primer: 5'-GCCAGGCTGGTCTCGAA-3', reverse primer: 5'-CTGAATAAATGAGTTCTGCAAAACAGGTT-3', VIC reporter probe: 5'-CTCAAGTGATTCACCCAC-3' and FAM reporter probe: 5'-CAAGTGATTCACCCAC-3'. Because of the low frequency of homozygotes for the variant allele, patient samples that were either heterozygous (TG) or homozygous (GG) for the variant allele were considered positive for the KRAS-variant.

Statistical analyses

Associations between the KRAS-variant and BRCA1-, BRCA2-, or non-BRCA1/BRCA2-related familial breast cancer, as well as associations between the KRAS-variant and specific characteristics of BRCA1 families were tested for significance by a 2 -test. Case-control odds ratios and their confidence intervals were calculated using Woolf approximations. The difference in mean age of onset between BRCA1 index cases carrying the variant allele and BRCA1 index cases not carrying the variant allele was determined by a t-test. The result of the Shapiro-Wilk test showed that the assumption of a normal distribution for the age of onset variable was valid. KRAS-variant frequencies among affected and unaffected BRCA1 carriers were compared using a Cox proportional hazard model. For this purpose, BRCA1 carriers were censored at either age of breast cancer diagnosis or age at last follow-up. Only BRCA1 carriers that were censored at breast cancer diagnosis were considered to be affected. Hazard ratios for the TG and GG genotypes combined were estimated using TT homozygotes as a baseline. P values of 0.05 or smaller were considered statistically significant. All statistical analyses were performed with STATA statistical package, release 11.0 (STATA Corp, College Station, TX).

Results

To establish whether the KRAS-variant (rs61764370 T>G) is implicated in breast cancer susceptibility, we have evaluated the prevalence of this variant in patients with breast cancer from BRCA1, BRCA2 and non-BRCA1/BRCA2 families as compared to controls. The variant allele was detected in 108 of 685 (15.8%) of indexes with breast cancer from non-BRCA1/BRCA2 families and 12 of 89 (13.5%) indexes with breast cancer from BRCA2 families. These frequencies were not significantly different from the 17.3% (138 of 797) prevalence among the control individuals. In contrast, the variant allele was present in 63 of 268 (23.5%) indexes with breast cancer from BRCA1 families, which was significantly different from the prevalence in controls (Table 1; P=0.025; OR=1.47 (1.05-2.06)).

However, after adjustment for multiple testing (Bonferroni correction for 3 tests) this difference was not significant anymore (P=0.073).

Because BRCA1 carriers mostly develop ER-negative breast cancers and BRCA2 carriers mostly develop ER-positive breast cancers, we wondered whether the KRAS-variant was associated with ER-negative breast cancer rather than mutant BRCA1 breast cancer. We had information on ER status of indexes from 483 non-BRCA1/BRCA2 families of whom 127 were ER-negative and 356 were ER-positive. The variant allele was present in 18 (14.2%) indexes with ER-negative breast cancer and 63 (17.7%) indexes with ER-positive breast cancer which was not significantly different from the prevalence in the controls (Table 1). These results suggest that the KRAS-variant not just simply associates with ER-negative breast cancer.

To evaluate whether the variant allele associates with a particular phenotype in BRCA1 families, we compared family characteristics of BRCA1 families with a KRAS-variant positive index case to BRCA1 families with a KRAS-variant negative index case (Table 2). There was no evidence for the variant to be associated with the presence of either one or more ovarian cancer cases or bilateral breast cancer cases in the family. We did find a higher frequency of the KRAS-variant in families with 3 or more breast cancer cases younger than 60 years compared with families with 2 or less breast cancer cases younger than 60 years (20.4% versus 27.9%), however this was not statistically significant (P=0. [START_REF] Oh | Hyperactivation of MAPK induces loss of ERalpha expression in breast cancer cells[END_REF]). Additionally, we found no difference in mean age of onset between BRCA1 index cases carrying the variant allele and BRCA1 index cases not carrying the variant allele (39.8 versus 39.0 years, respectively; P=0.55).

In order to gain more statistical power for the analysis of association between the KRASvariant and mutant BRCA1 breast cancer, we expanded this analysis by including other family members in addition to the index cases. In total, we had DNA from 390 BRCA1 carriers with breast cancer available for genotyping among the 268 BRCA1 families. However, the KRAS-variant was present in only 20.3% (79 of 390) of affected BRCA1 carriers (Table 3), which is lower than the initial 23.5% we found among the index cases (Table 1). Consequently, the prevalence of the KRAS-variant among BRCA1 carriers was not significantly different from the controls (P=0.22; OR=1.21 (0.89-1.65)), despite expansion of the number of BRCA1 carriers with breast cancer.

To determine whether or not the KRAS-variant modifies BRCA1 mutant breast cancer risk, all affected and unaffected BRCA1 carriers among the 268 BRCA1 families were genotyped for the variant allele. Mean age at censure was 41.0 years for affected BRCA1 carriers and 45.5 years for unaffected BRCA1 carriers. Similar to the frequency in the controls, the KRAS-variant was present in 17.3% (45 of 260) unaffected BRCA1 carriers (Table 3). Importantly, there was no evidence of association between the KRAS-variant and breast cancer risk among BRCA1 carriers (Table 3; P=0.50; HR=1.07 (0.88-1.30)).

Discussion

In the current study we have found an increase in the frequency of the KRAS-variant (rs61764370 T>G) in breast cancer index cases from BRCA1 families as compared to geographically-matched controls, whereas the frequency of this variant allele was not significantly different among breast cancer index cases from BRCA2 or non-BRCA1/BRCA2 families. However, expansion of the number of BRCA1 carriers with breast cancer by including other family members in addition to the index cases did not improve significance for the association between the variant allele and mutant BRCA1 breast cancer. Additionally, the KRAS-variant did not appear to modify breast cancer risk for BRCA1 carriers.

Still the question remains whether the breast cancer risk conferred by the KRAS-variant is not too small to be detected in our case-control study. Considering the relatively high prevalence in the general population, the variant allele would not be expected to confer a dramatically increased breast cancer risk. Also, the breast cancer risk conferred by the KRAS-variant may be larger in GG homozygotes than TG heterozygotes. Therefore, we feel that this trend towards association between the KRAS-variant and mutant BRCA1 breast cancer requires validation in a larger cohort of BRCA1 carriers, providing more statistical power and enabling the analysis of TG heterozygotes and GG homozygotes separately.

Interestingly, stratification of breast cancer patients from non-BRCA1/BRCA2 families according to their tumor's ER-status did not reveal an increased KRAS-variant frequency among ERnegative breast cancers, suggesting that the variant is not just simply associated with ER-negative breast cancer. This may seem unexpected as the KRAS-variant has been shown to lead to increased KRAS expression in vitro and hyperactivation of the MAPK pathway has been shown to downregulate ER expression in vitro [START_REF] Chin | A SNP in a let-7 microRNA complementary site in the KRAS 3' untranslated region increases non-small cell lung cancer risk[END_REF][START_REF] Oh | Hyperactivation of MAPK induces loss of ERalpha expression in breast cancer cells[END_REF]. However, one may wonder whether the level of increased KRAS expression as a result of the KRAS-variant is similar to that of an oncogenic RAS or RAF mutation. This may thus be a matter of dosage and therefore MAPK pathway activation should be assessed in tumors from patients that carry the KRAS-variant. Unfortunately, due to missing ERBB2 status of the vast majority of non-BRCA1/BRCA2 cases we have not been able to assess variant allele frequencies in index cases with triple (ER, PR, and ERBB2) negative breast cancers, preventing us to study whether the KRAS-variant frequency is increased specifically in BRCA1 carriers or more generally in patients with triple negative or basal-like breast cancer.

If the KRAS-variant were to be specifically associated with mutant BRCA1 breast cancer, this would suggest a biological interaction between BRCA1 and KRAS or between BRCA1 and let-7 miRNA. Many human cancers, including breast cancers, display altered expression of the let-7 miRNA family and let-7 has been shown to act as a tumor suppressor regulating the expression of RAS, HMGA2, LIN28 and PEBP1 [START_REF] Esquela-Kerscher | Oncomirs -microRNAs with a role in cancer[END_REF][START_REF] Day | Lal A MicroRNAs and their target gene networks in breast cancer[END_REF][START_REF] Johnson | RAS is regulated by the let-7 microRNA family[END_REF]. Recently, let-7 was shown to regulate stem cell-like properties in breast tumor initiating cells as downregulation of let-7 increased proliferation and selfrenewal, whereas expression of let-7 enhanced differentiation [START_REF] Yu | let-7 regulates self renewal and tumorigenicity of breast cancer cells[END_REF]. Because the KRAS-variant allele was associated with lower let-7 levels in NSCLC patients as compared to patients without the variant allele, the KRAS-variant may therefore act in tumorigenesis through maintaining a proliferative cell state [START_REF] Chin | A SNP in a let-7 microRNA complementary site in the KRAS 3' untranslated region increases non-small cell lung cancer risk[END_REF]. Interestingly, knock-down of BRCA1 in primary breast epithelial cells was shown to increase the number of cells displaying a stem/progenitor phenotype [START_REF] Liu | BRCA1 regulates human mammary stem/progenitor cell fate[END_REF]. The presence of the KRAS-variant in the germline of a BRCA1 carrier may thus further facilitate this stem cell phenotype imposed by BRCA1. If the KRAS-variant would eventually be conclusively associated with mutant BRCA1 breast cancer, it might be interesting to study this hypothesis in future research. miRNAs make up one of the most abundant classes of regulatory genes, with 30% of the human genes harboring miRNA target sites [START_REF] Lewis | Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets[END_REF][START_REF] Rajewsky | microRNA target predictions in animals[END_REF]. Thus far, many miRNAs have already been implicated in cancer as either oncogenes or tumor suppressor genes [START_REF] Esquela-Kerscher | Oncomirs -microRNAs with a role in cancer[END_REF][START_REF] Day | Lal A MicroRNAs and their target gene networks in breast cancer[END_REF]. This makes both the miRNA genes themselves as well as the miRNA binding sites in the 3'UTR of well-known cancer genes attractive candidate breast cancer susceptibility alleles. Although several key targets of many miRNAs have been identified, also many target genes remain elusive. A significant portion of these miRNA target genes include well-known breast cancer oncogenes and tumor suppressor genes, such as ERBB2, CCND1, MYC and PTEN, but also breast cancer susceptibility genes such as ATM [START_REF] Day | Lal A MicroRNAs and their target gene networks in breast cancer[END_REF][START_REF] Hu | ATM is down-regulated by N-Myc-regulated microRNA-421[END_REF]. The KRAS-variant was the first single nucleotide variant in a miRNA binding site shown to be implicated in cancer risk, but many more are likely to be discovered. Screening of miRNA genes and their target sequences in the 3'UTR of genes known to be implicated in breast cancer may thus provide a productive strategy for defining part of the excess familial breast cancer risk. -test. Case-control odds ratios and their confidence intervals were calculated using Woolf approximations. *After adjustment for multiple testing (Bonferroni correction for 3 tests) the P-value between BRCA1 carriers and controls was not statistically significant anymore (P=0.073).

Table 1

 1 KRAS 3'UTR SNP genotype frequencies among indexes from BRCA1, BRCA2 and non-

	BRCA1/BRCA2 families
	Population	Genotype Frequency	OR	95% CI	P value
	Controls	GG+TG	138/797 (17.3%)	1.00
		TT	659/797 (82.7%)
	BRCA1 carriers	GG+TG	63/268 (23.5%)	1.47 1.05-2.06 0.025*
		TT	205/268 (76.5%)
	BRCA2 carriers	GG+TG	12/89 (13.5%)	0.74 0.39-1.40 0.36
		TT	77/89 (86.5%)
	Non-BRCA1/BRCA2 carriers GG+TG	108/685 (15.8%)	0.89 0.68-1.18 0.42
		TT	577/685 (84.2%)
		ER-positive GG+TG	63/356 (17.7%)	1.03 0.74-1.43 0.87
		TT	293/356 (82.3%)
	ER-negative GG+TG	18/127 (14.2%)	0.79 0.46-1.34 0.38
		TT	109/127 (85.8%)
	Differences in KRAS-variant allele frequencies between cases and controls were tested for
	significance by a	2

Table 2

 2 Association of the KRAS-variant with specific characteristics of BRCA1 families Case-control odds ratios and their confidence intervals were calculated using Woolf approximations. OvC, ovarian cancer; Bil, bilateral; BrC, breast cancer. Table3KRAS 3'UTR SNP genotype frequencies among affected and unaffected BRCA1 carriers Differences in KRAS-variant allele frequencies between affected and unaffected BRCA1 carriers were tested for significance by a Cox proportional hazards model. Mean age at censure was 41.0 years for affected BRCA1 carriers and 45.5 years for unaffected BRCA1 carriers. Hazard ratios for the TG+GG genotype were estimated using TT homozygotes as a baseline.

	Family characteristic	Frequency of the	OR	95% CI	P value
			GG+TG genotype
	No OvC in family	38/152 (25.0%)
	One or more OvC in family	22/96 (22.9%)	0.89 0.49-1.63 0.71
	No Bil BrC in family	36/147 (24.5%)
	One or more Bil BrC in family	24/106 (22.6%)	0.90 0.50-1.63 0.73
	Less than 3 BrC < 60 years in family	29/142 (20.4%)
	3 or more BrC < 60 years in family	31/111 (27.9%)	1.51 0.84-2.70 0.16
	Association of the KRAS-variant with specific characteristics of BRCA1 families was tested for
	significance by a
	Genotype Frequency in	Frequency in carriers	HR	95% CI	P value
		unaffected carriers	with breast cancer
	TT	215/260 (82.7%)	311/390 (79.7%)	1.00
	GG+TG	45/260 (17.3%)	79/390 (20.3%)	1.07 0.88-1.30 0.50

2

-test.

Acknowledgements

We wish to thank all members of the breast cancer families that have participated in this study. We also thank Renée Foekens, Bahar Özturk, Marc Kribbe and Rahena van Kampen-Binda for assistance with collecting DNA samples. Financial support was provided by the Netherlands Genomics Initiative (NGI)/Netherlands Organization for Scientific Research (NWO), a K08 grant from the NIH [CA124484] and a CTSA Grant, Number UL1 RR024139 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), and NIH roadmap for Medical Research.

Conflict of interest statement

No authors have a conflict of interest with the content of this manuscript.


Manuel Koch

Fadi Hussein

Andreas Woeste

Carsten Gründker

Karl Frontzek

Günter Emons

M.D. Thomas Hawighorst

CD36-mediated activation of endothelial

Introduction

There is large amount of evidence that tumor growth is dependent on angiogenesis [START_REF] Carmeliet | Angiogenesis in cancer and other diseases[END_REF][START_REF] Ferrara | Angiogenesis as a therapeutic target[END_REF][START_REF] Folkman | Tumor angiogenesis: therapeutic implications[END_REF]. Clinically, proof of concept has begun with the recent regulatory approvals of three antiangiogenic therapies targeting the vascular endothelial growth factor (VEGF) pathway.

Despite early benefits seen in breast cancer patients treated with anti-VEGF therapies [START_REF] Miller | Paclitaxel plus bevacizumab versus paclitaxel alone for metastatic breast cancer[END_REF],

enduring efficacy with regard to survival benefits are relatively modest [START_REF] Kerbel | Tumor angiogenesis[END_REF]. In addition, recent data provided strong evidence that tumors may escape or become resistant to the antiangiogenic effects of blocking the VEGF signaling pathway [START_REF] Ebos | Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis[END_REF][START_REF] Paez-Ribes | Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis[END_REF]. Therefore, additional antiangiogenic approaches need to be evaluated. Two members of the thrombospondin (TSP) family [START_REF] Adams | The thrombospondins[END_REF][START_REF] Kazerounian | Thrombospondins in cancer[END_REF], TSP-1 and TSP-2, are important naturally occurring angiogenesis inhibitors. Both proteins were shown to have potent antiangiogenic activity by inhibiting endothelial cell proliferation, migration, and tube formation in response to multiple angiogenic stimuli [START_REF] Lawler | The functions of thrombospondin-1 and-2[END_REF]50],

and by inhibiting angiogenic responses in a number of in vivo models [START_REF] Adams | The thrombospondins[END_REF]. The antiangiogenic activity of TSP-1 was localized to the properdin-like type I repeats, also called thrombospondin structural homology repeats (TSR), in the N-terminal domain of TSP-1 [START_REF] Iruela-Arispe | Inhibition of angiogenesis by thrombospondin-1 is mediated by 2 independent regions within the type 1 repeats[END_REF]. In contrast to TSP-1, the molecular mechanism of the antiangiogenic activity of TSP-2 has not been as well studied. Since it has been reported that TSP-2 is more effective in inhibiting tumor growth and angiogenesis than TSP-1 [START_REF] Kyriakides | Mice that lack thrombospondin 2 display connective tissue abnormalities that are associated with disordered collagen fibrillogenesis, an increased vascular density, and a bleeding diathesis[END_REF] [28] [START_REF] Streit | Thrombospondin-2: a potent endogenous inhibitor of tumor growth and angiogenesis[END_REF], we generated a recombinant fusion protein consisting of the N-terminal region of TSP-2 and an IgG-Fc1 fragment (N-TSP2-Fc). N-TSP2-Fc contained the procollagen homology domain as well as three type I repeats of TSP-2. Here, we report that recombinant N-TSP2-Fc induced apoptosis of endothelial cells by CD36 receptor mediated loss of mitochondrial membrane potential and activation of caspase-3. In contrast to the majority of experimental antiangiogenic studies that only focused on the effects on primary tumor growth, with much less attention on metastasis, we provide strong evidence that N-TSP2-Fc significantly inhibited tumor growth of xenotransplanted MDA-MB-435 and MDA-MB-231 breast cancer cells and both lymph node as well as lung metastasis of MDA-MB-435 tumor cells grown as xenotransplants in the mammary gland of nude mice.

Materials and Methods

Cell lines and culture. Human MDA-MB-435 and MDA-MB-231 breast cancer cells were obtained from ATCC and maintained in DMEM medium (Invitrogen) containing 10% FCS, 4.5 mg/ml glucose, 2mM L-glutamine, 100 units/ml penicillin G, and 100 µg/ml streptomycin.

Human 293-EBNA (Epstein-Barr virus nuclear antigen) cells (Invitrogen) were maintained in DMEM/F12 medium (Invitrogen) containing 10% FCS, 15 mM HEPES, 1.0 µg/ml puromycin, 100 units/ml penicillin G, 100 µg/ml streptomycin, and 0.25 µg/ml fungizone, amphotericin B (Invitrogen). Human dermal microvascular endothelial cells (HDMEC) from Promocell (Heidelberg, Germany) were cultured in ECGM-MV medium (Promocell) containing fetal calf serum (0.05 ml/ml), endothelial cell growth supplement (0.004 ml/ml), recombinant human epidermal growth factor (10 ng/ml), heparin (22.5 µg/ml), hydrocortisone (1 µg/ml), and phenol red (0.62 ng/ml) supplemented with 50 U/ml penicillin and 50 µg/ml streptomycin.

Cell transfection.

The coding sequences of the N-terminal region of TSP-2 (aa 18-550 of human TSP-2) and the Fc region of human IgG1 (hinge and CH" and CH3 domains) were obtained by PCR and cloned into the the NheI and BamHI sites of a modified pCEP4 expression vector [START_REF] Koch | A novel member of the netrin family, beta-netrin, shares homology with the beta chain of laminin: identification, expression, and functional characterization[END_REF] that contains a cytomegalovirus enhancer/promoter, the EBNA-1 gene, and a puromycin selection cassette. Human 293-EBNA cells were stably transfected with the expression vector using the Fugene transfection reagent (Roche).

Purification of recombinant N-TSP2-Fc. The secreted fusion protein was purified from serum-free culture medium by using two-step affinity chromatography with Gelatin Sepharose 4B and Protein G Sepharose (GE Healthcare Europe). The fusion protein was eluted with 0.1M glycine (pH 2.7), and each fraction was neutralized with 1M Tris-HCl (pH 9.0). After dialysis the protein was stored at -80 0 C. The protein concentration was determined using the BCA protein assay kit (Pierce).

Tube formation assay. Twenty-four-well plates were coated with 300 µl Matrigel (BD Pharmingen) and incubated for 1h at 37 0 C. HDMEC (1 x 10 5 cells) in ECGM-MV containing VEGF (20 ng/ml) were seeded into duplicate and observed at 12h. The formation of tube-like structures was analyzed using a Olympus IX51 microscope. Images were captured and computer-assisted analyses of tube length were performed using the Photoshop CS3 software (Adobe Systems).

Apoptosis assay. Primary HDMEC incubated in ECGM containing VEGF 165 (R&D Systems) at a concentration of 20 ng/ml were incubated with 40 µg/ml of N-TSP2-Fc with or without concurrent addition of 10 µg/ml of blocking CD36 antibody (Abcam) for 72h. Apoptosis was evaluated by using a procedure similar to that described by Nicoletti et al. [START_REF] Nicoletti | A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry[END_REF]. Briefly, a pellet containing 1x10 6 HDMEC was gently resuspended in 500 mL of hypotonic fluorochrome solution containing 0.1% Triton X-100 (Sigma), 0.1% sodium citrate, and 50 mg/mL propidium iodide (Sigma). After overnight incubation the cell suspensions were assayed by flow cytometry analysis of cellular DNA content on a FACSCalibur equipment (Becton Dickinson Immunocytometry Systems,) using CellQuest software (Becton Dickinson Immunocytometry Systems).

Mitochondrial membrane potential measurement. HDMEC cultured in ECGM containing

VEGF 165 (R&D Systems) at a concentration of 20 ng/ml were incubated with 40 µg/ml of N-TSP2-Fc with or without concurrent addition of 10 µg/ml of blocking CD36 antibody (Abcam) for 72h. The mitochondrial membrane potential was analyzed using the JC-1 mitochondrial membrane potential detection kit (Biotium).

Western blot analysis of caspase-3 activity. HDMEC were treated with N-TSP2-Fc (final concentration 40 µg/ml) with or without concurrent addition of 10 µg/ml of blocking CD36 antibody (Abcam) for 72h. Cell lysates from HDMEC were separated on SDS/PAGE under reducing conditions and transferred to nitrocellulose membranes (HybonD-ECL, GE Healthcare Europe, Munich, Germany) and then immunoblotted with a rabbit anti-human active caspase-3 polyclonal antibody (BD Pharmingen). The specifically bound antibody was detected using the enhanced chemiluminescence kit (Millipore). The bands were analyzed using the Kodak image system (Kodak).

Tumorigenesis and metastasis assay.

2×10 6 MDA-MB-435 or 5×10 6 MDA-MB-231 breast cancer cells were injected bilaterally into the second mammary fat pads of athymic, female, 6-wk-old NCR nu/nu mice. The tumor volumes were calculated using the following formula: Volume=4/3x x(1/2xsmaller diameter) 2 x1/2xlarger diameter. After sacrifying the mice, tumors, axillary lymph nodes and lungs were removed from each mouse. The tissue was embedded in OCT compound or was snap frozen in liquid nitrogen. All animal studies were done according to the German ethical guidelines and the German laws for protection of animals.

Inhibition ELISA. Mice were intraperitoneally injected with N-TSP2-Fc (100 µg /100 µl PBS).

Serum samples were obtained at various times (0h, 0,5h, 1h, 2h, 6h, 12h, and 24h) after injection. The concentration of TSP-2 was determined using an inhibition-ELISA. 96-well plates (MaxiSorbTM, Nunc) were coated overnight with N-TSP2-Fc at a concentration of 1 µg/mL. The remaining reactive sites were blocked with 1% BSA. A polyclonal goat antibody directed against the N-terminal region of human TSP-2 (R&D) was previously mixed in 96-well plates either with standard solutions of N-TSP2-Fc (ranging from 2 pg/mL to 20 µg/mL) or with mouse serum to be tested that was diluted 1:50 and 1:500 with PBS. This mixture was added to the N-TSP2-Fc-coated wells and incubated for 2h at RT. The samples were assayed using a chemiluminescent substrate (SuperSignal ELISA Pico substrate). Luminescence was measured at 425nm using a multi-detection microplate reader (BioTek Instruments GmbH).

Data were analyzed using the SigmaPlot software (Systat Software) and the concentration of TSP-2 in each sample was calculated from the standard curve.

Analysis of tumor angiogenesis.

Cryostat sections (5 µm) were stained with a rat anti-mouse CD31 monoclonal antibody (BD Pharmingen). Representative sections obtained from five tumors were analyzed using a Olympus IX51 microscope. Images were captured and morphometric analyses were performed using the IP LAB software (Scanalytics) as described [START_REF] Streit | Thrombospondin-2: a potent endogenous inhibitor of tumor growth and angiogenesis[END_REF]. Three different fields in each section were examined at 10x magnification, and the number of vessels per mm 2 , the vessel size, and the relative area occupied by tumor blood vessels within the tumors were determined.

Quantitative analysis of metastasis.

The DNEasy Blood and Tissue Kit (Qiagen) was used for isolation of genomic DNA. Primers (Eurogente) specific for the human alu sequences (sense: 5'-CATGGTGAAACCCCGTCTCTA -3'; and antisense: 5'-GCCTCAGCCTCCCGAGTAG-3') were used to amplify human alu repeats present in genomic DNA that was extracted from mouse tissue. The internal TaqMan probe for alu (5'-[YY]-ATTAGCCGGGCGTGGTGGC-[BHQ-1]-3') was 5`-labelled with the reporter fluorescent dye Yakima Yellow (YY) and carried the non-fluorescent quencher dye Black Hole Quencher-1 (BHQ-1). The 18S rRNA control kit (Eurogentec) was used as an internal reference and was labelled with the reporter dye 6-carboxy-fluorescein (FAM) at the 5´end and the quencher dye 6-carboxy-tetramethyl-rhodamin (TAMRA) at the 3´end. PCRs were carried out in 25 µl using 2xTaqMan Universal PCR Master Mix (Applied Biosystems), 0.9µM each alu specific oligonucleotide primer, 250nM internal alu probe, 1.5 µl 18S rRNA primer mix, 0.5 µl internal 18S rRNA probe, and 1 µl genomic DNA. The final concentration for genomic DNA was 5ng/µl for lymph node tissue and 50 ng/µl for lung tissue. PCR was carried out for 2min at 50°C for 10min at 95°C followed by 40 cycles of 15s at 95 0 C and 1min at 60 0 C using the ABI PRISM™ 7700 Sequence Detector (Applied Biosystems). Serial dilution steps (1:10) of genomic DNA obtained from human MDA-MB-435 cells in genomic DNA obtained from either mouse lymph node or lung tissue served to construct the calibration curves. The cycle at which the alu PCR product becomes detectable above background signal (Ct alu ) was determined for each dilution series. The alu signal was normalized against the relative quantity of 18S rRNA and expressed as ∆Ct=Ct alu -Ct 18SrRNA . A standard curve was generated for each measurement by using the real-time PCR assay and by plotting ∆Ct as a function of the relative amount of human DNA in murine DNA (%). The R 2 value (correlation coefficient) was >0.96 for all measurements demonstrating an excellent relationship between the alu signal and the relative amount of human DNA with a detection limit of 0.001% human DNA in murine DNA.

Statistical analysis

The two-sided unpaired t-test was used to analyze the tumor and metastasis data and to analyze the differences in microvessel density, vessel size, and total vascular area and total vascular area. Tube formation, apoptosis, mitochondrial membrane potential, and caspase-3 activity were analyzed for significant differences by one-way ANOVA followed by Student-Newman-Keuls`test for comparison of individual groups, after a Bartlett test had shown that variances were homogenous.

Results

N-TSP2-Fc inhibits tube formation of CD36 positive endothelial cells. Puromycin-selected human 293-EBNA cell clones secreting high amounts of N-TSP2-Fc were used for large-scale expression. The molecular weight of the purified recombinant protein was determined as approximately 110kDa by SDS-PAGE followed by western blot (data not shown). N-terminal amino acid sequence analysis confirmed that the recombinant protein included amino acid 18 to 550 of human TSP-2 containing the heparin-binding-domain, the procollagen-like domain and the three type 1 (properdin-like) repeats. The antiangiogenic efficacy was analyzed by investigating the effect of N-TSP2-Fc on VEGF-induced tube formation of HDMEC cultured on matrigel. After 12h, formation of networks with interconnection of tube-like structures was detected in HDMEC culture (Fig. 1a). In contrast, only sparse tube-like structures were detected when HDMEC were treated with N-TSP2-Fc (Fig. 1c) resulting in a significant reduction of the average tube length by more than 50% (Fig. 1e). After incubation of HDMEC with blocking CD36 antibody addition of N-TSP2-Fc resulted in tube formation that was comparable to untreated control (Fig. 1d and1e) demonstrating that activation of the CD36 receptor was involved in the pronounced inhibition of network formation by N-TSP2-Fc.

Treatment of HDMEC with IgG-Fc did not show any significant effect on VEGF-induced network formation providing evidence that the Fc fragment is not involved in the antiangiogenic activity of N-TSP2-Fc (Fig. 1b and1e).

N-TSP2-Fc induces activation of apoptosis in microvascular endothelial cells. Flow

cytometry of the sub-G 1 apoptotic population showed that treatment of HDMEC with N-TSP2-Fc for 72h in the presence of VEGF resulted in a significant increase of apoptotic endothelial cells by more than 45% as compared to control treated cells (Fig. 2a). Parallel incubation with blocking CD36 antibody attenuated N-TSP2-Fc-induced apoptosis in HDMEC to close to the basal level indicating that CD36 is required for the apoptotic activity of N-TSP2-Fc (Fig. 2a).

N-TSP2-Fc causes activation of caspase-3 and of the mitochondrial-dependent intrinsic pathway of apoptosis.

Treatment of HDMEC with N-TSP2-Fc for 72h resulted in a significant loss of mitochondrial membrane potential and decreased the mitochondrial membrane potential to 66% of control (=100%; P < 0.001). When HDMEC were treated with CD36 blocking antibody, addition of N-TSP2-Fc failed to induce loss of mitochondrial membrane potential (Fig. 2b). In addition, treatment of HDMEC with N-TSP2-Fc for 72h significantly induced activation of caspase-3 by more than 100% as compared to PBS control (Fig. 2c and2d). The activation of caspase-3 by N-TSP2-Fc was almost completely abrogated by parallel incubation of HDMEC with blocking CD36 antibody (Fig. 2c and2d).

Systemic treatment with N-TSP2-Fc inhibits tumor growth and angiogenesis. MDA-MB-

435 tumor cells were subcutaneously injected into the second mammary gland of nude mice.

Six days after tumor cell injection mice received either vehicle control (PBS), IgG-Fc (5 mg/kg/d), or N-TSP2-Fc (5 mg/kg/d). Control treated mice formed tumors reaching a volume of ~600 mm 3 within 21 days (Fig. 1S). Following systemic treatment with N-TSP2-Fc for 15 days, MDA-MB-435 tumors reached a volume of ~220 mm 3 , representing a 63% inhibition of tumor growth (P < 0.001; Fig. 1S). At this time point, treatment with IgG-Fc did not result in any major difference of tumor growth as compared to control mice (P = 0.39; Fig. 1S). We next investigated whether the prolongation of the growth period might change the inhibitory effect on tumor growth by N-TSP2-Fc (5 mg/kg body weight) treatment. After a growth period of 36 days the experiment had to be terminated due to tumor burden and first signs of tumor ulceration in the control group according to the institutional regulations on animal experiments. Systemic treatment was initiated 6 days after tumor cell injection and continued daily for 30 days. The inhibitory effect of N-TSP2-Fc became highly significant at day 22 of MDA-MB-435 tumor growth and was maintained until the end of the growth period resulting in a 61% inhibition of tumor growth at day 36, compared to control-treated tumors (P < 0.001; Fig 3a). To analyze tumor-associated vascularization frozen sections of MDA-MB-435 tumors were stained for the endothelial junction molecule CD31 [START_REF] Dejana | Endothelial cell-to-cell junctions[END_REF]. Computer-assisted morphometric image analysis revealed that both the vessel density (Fig. 3b) and the average vessel size (Fig. 3c) was significantly decreased in N-TSP2-Fc treated tumors, as compared with control tumors, resulting in a significant decrease in the relative area occupied by tumor vessels by 67% (P < 0.001; Fig. 3d). To determine the serum concentration of both TSP-2 and N-TSP2-Fc we established an inhibition ELISA using an antibody directed against the N-terminal region of TSP-2. The serum concentration of TSP-2 in untreated tumor-bearing mice was 28.4±3.6 ng/ml (Table 1). Intraperitoneal administration of N-TSP2-Fc resulted in an increase of the serum concentration of TSP-2 and this effect lasted up to 24h. Since intraperitoneal injection of PBS did not result in any major differences in the serum concentration of TSP-2 (data not shown) the observed higher level of serum TSP-2 is due to an increase of the concentration of recombinant N-TSP2-Fc. As early as 30 minutes after intraperitoneal injection we found a strong increase in the serum level of N-TSP2-Fc (Table 1) that reached a peak at 1h after intraperitoneal injection. Afterwards the serum concentration of N-TSP2-Fc rapidly decreased at 12h after injection and reached a serum concentration that was close to the TSP-2 level of untreated tumor-bearing mice at 24h after intraperitoneal administration (Table 1). In addition, we examined the effect of recombinant N-TSP2-Fc on in vivo tumor growth of orthotopically injected MDA-MB-231 breast cancer cells (Fig. 2S). One week after tumor cell injection, mice were treated daily either with N-TSP2-Fc (5 mg/kg body weight) or vehicle control. Four weeks after tumor cell injection systemic treatment with N-TSP2-Fc resulted in a significant inhibition of xenotransplanted MDA-MB-231 breast carcinomas as compared to control treated tumors (P < 0.05; Fig. 2S).

Systemic treatment with N-TSP2-Fc decreases metastasis.

Results of alu PCR indicated the presence of human DNA as early as two weeks after tumor cell injection in both lymph node and lung tissue of tumor bearing mice treated with vehicle control (Fig. 4a and4b). The relative amount of human DNA in murine DNA at this time point was 0.41% in lymph node tissue and 0.024% in lung tissue of tumor bearing mice as compared to the background signal of 7.4x10 -4 % and 4.2x10 -3 % in non-tumor bearing mice, respectively (Fig. 4a and4b). Treatment of tumor-bearing mice with N-TSP2-Fc resulted in a significant reduction of both lymph node and lung metastasis at two weeks after tumor implantation (Fig. 4a and4b). At five weeks after tumor cell injection the relative amount of human tumor DNA was reduced by 80% in lymph node tissue and by 68% in lung tissue of N-TSP2-treated mice as compared to control mice (Fig. 4a and4b).

Discussion

TSP-2 is a potent endogenous inhibitor of tumor growth and angiogenesis [START_REF] Hawighorst | Thrombospondin-2 plays a protective role in multistep carcinogenesis: a novel host anti-tumor defense mechanism[END_REF][START_REF] Streit | Thrombospondin-2: a potent endogenous inhibitor of tumor growth and angiogenesis[END_REF].

The antitumoral effect of TSP-2 is stronger than the effect of TSP-1 [START_REF] Streit | Thrombospondin-2: a potent endogenous inhibitor of tumor growth and angiogenesis[END_REF]. However, the exact molecular mechanism of the antiangiogenic activity of TSP-2 has not been as well studied as that of TSP-1. TSP-2 shows only low sequence homology to TSP-1 in the procollagen region.

Moreover, a recombinant fragment of human TSP-2 that only included the N-terminal globular domain and the procollagen homology domain failed to significantly inhibit tumor growth and angiogenesis [START_REF] Noh | An N-terminal 80 kDa recombinant fragment of human thrombospondin-2 inhibits vascular endothelial growth factor induced endothelial cell migration in vitro and tumor growth and angiogenesis in vivo[END_REF]. Therefore, the antiangiogenic activity of TSP-2 might map to the properdinlike type 1 repeats [START_REF] Tolsma | Peptides derived from two separate domains of the matrix protein thrombospondin-1 have antiangiogenic activity[END_REF], which have been implicated in the antiangiogenic activity of TSP-1 [START_REF] Dawson | Three distinct D-amino acid substitutions confer potent antiangiogenic activity on an inactive peptide derived from a thrombospondin-1 type 1 repeat[END_REF][START_REF] Iruela-Arispe | Inhibition of angiogenesis by thrombospondin-1 is mediated by 2 independent regions within the type 1 repeats[END_REF][START_REF] Vogel | Modulation of endothelial cell proliferation, adhesion, and motility by recombinant heparin-binding domain and synthetic peptides from the type I repeats of thrombospondin[END_REF]. The antiangiogenic effects within the properdin-like type 1 repeats of TSP-1 are mediated partly through interaction of the CSVTCG sequence with the CD36 receptor on endothelial cells [START_REF] Dawson | CD36 mediates the In vitro inhibitory effects of thrombospondin-1 on endothelial cells[END_REF][START_REF] Jimenez | Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1[END_REF][START_REF] Tolsma | Peptides derived from two separate domains of the matrix protein thrombospondin-1 have antiangiogenic activity[END_REF].

We produced a recombinant fusion protein, N-TSP2-Fc, that encompasses the Nterminal globular region with all three type 1 repeats containing two CSVTCG sequence motifs within the first and second type 1 repeats. N-TSP2-Fc exerted potent antiangiogenic activity in vitro. We found that N-TSP2-Fc significantly inhibited HDMEC tube formation and induced HDMEC apoptosis. N-TSP2-Fc also resulted in a significant increase of caspase-3 activity and a significant loss of mitochondrial membrane potential in cultured HDMEC. Moreover, blockade of the CD36 receptor almost completely abrogated the reported in vitro effects of N-TSP2-Fc, strongly suggesting that the antiangiogenic activity of N-TSP2-Fc is mediated by CD36dependent induction of endothelial cell apoptosis and at least in part through activation of the intrinsic apoptotic pathway. This is in agreement with the previously reported binding of TSP-2 to CD36 and lack of inhibition of angiogenesis by TSP-2 in CD36 null mice [START_REF] Simantov | The antiangiogenic effect of thrombospondin-2 is mediated by CD36 and modulated by histidine-rich glycoprotein[END_REF]. The exact molecular mechanisms through which TSP-2 and TSRs induce activation of caspase-3 and promote CD36-mediated apoptosis remain to be elucidated.

We investigated the efficacy of systemic therapy with N-TSP2-Fc on both tumor growth and metastasis. To this end we made use of MDA-MB-435 tumor cells representing a well established orthotopic model system for the study of breast cancer metastasis. MDA-MB-435 cells form primary tumors when injected into the mammary fat pad of nude mice, and spontaneously metastasize from these tumors to the lymph nodes and lungs [START_REF] Price | Tumorigenicity and metastasis of human breast carcinoma cell lines in nude mice[END_REF][START_REF] Skobe | Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis[END_REF][START_REF] Welch | Technical considerations for studying cancer metastasis in vivo[END_REF].

However, in a microarray expression study MDA-MB-435 cells have been reported to cluster with several melanoma cell lines [START_REF] Ross | Systematic variation in gene expression patterns in human cancer cell lines[END_REF] and it has been speculated that that MDA-MB-435 cell is instead the M14 melanoma cell line, due to an early cross-contamination of cell cultures [START_REF] Chambers | MDA-MB-435 and M14 cell lines: identical but not M14 melanoma?[END_REF][START_REF] Rae | MDA-MB-435 cells are derived from M14 melanoma cells--a loss for breast cancer, but a boon for melanoma research[END_REF]. [START_REF] Sellappan | Lineage infidelity of MDA-MB-435 cells: expression of melanocyte proteins in a breast cancer cell line[END_REF] as well as epithelial markers such as cytokeratin 19 and secrete milk proteins and lipids [START_REF] Sellappan | Lineage infidelity of MDA-MB-435 cells: expression of melanocyte proteins in a breast cancer cell line[END_REF] suggesting that MDA-MB-435 breast cancer cells may have undergone a lineage infidelity during tumor progression [START_REF] Sellappan | Lineage infidelity of MDA-MB-435 cells: expression of melanocyte proteins in a breast cancer cell line[END_REF]. Indeed, lineage infidelity has been seen in breast carcinomas that express both epithelial and melanoma-associated proteins [START_REF] Nobukawa | Breast carcinoma diverging to aberrant melanocytic differentiation: a case report with histopathologic and loss of heterozygosity analyses[END_REF][START_REF] Padmore | Primary combined malignant melanoma and ductal carcinoma of the breast. A report of two cases[END_REF]. Very recently, Chambers [START_REF] Chambers | MDA-MB-435 and M14 cell lines: identical but not M14 melanoma?[END_REF] reported that although currently available stocks of the MDA-MB-435 and M14 cell lines were shown to be virtually identical at the karyotype level [START_REF] Rae | MDA-MB-435 cells are derived from M14 melanoma cells--a loss for breast cancer, but a boon for melanoma research[END_REF], the conclusion that M14 and MDA-MB-435 cells are of M14 melanoma origin is inconsistent with the fact that the M14 cell line derived from a male patient with metastatic melanoma [START_REF] Chee | Production of melanoma-associated antigen(s) by a defined malignant melanoma cell strain grown in chemically defined medium[END_REF] and the female karyotypes of both cell lines at present [START_REF] Chambers | MDA-MB-435 and M14 cell lines: identical but not M14 melanoma?[END_REF][START_REF] Rae | MDA-MB-435 cells are derived from M14 melanoma cells--a loss for breast cancer, but a boon for melanoma research[END_REF].

MDA-MB-435 cells have been shown to express melanocytic proteins

Chambers [START_REF] Chambers | MDA-MB-435 and M14 cell lines: identical but not M14 melanoma?[END_REF] suggested that the evidence from the literature rather is consistent with cell lines being of MDA-MB-435 breast cancer origin and that the MDA-MB-435 cell line represents a poorly differentiated aggressive breast tumor line with expression of both epithelial and melanocytic markers. In addition, in a recent study on distinct gene mutation profiles among luminal-type and basal-type breast cancer cell lines Hollestelle et al. [START_REF] Hollestelle | Distinct gene mutation profiles among luminal-type and basal-type breast cancer cell lines[END_REF] revealed that the MDA-MB-435 cell line was basal-type. They identified subtype-specific gene mutation profiles revealing that basal-type breast cancers may have a genetic basis similar to melanomas [START_REF] Hollestelle | Distinct gene mutation profiles among luminal-type and basal-type breast cancer cell lines[END_REF][START_REF] Hollestelle | Comment Re: MDA-MB-435 and M14 cell lines: identical but not M14 Melanoma?[END_REF]. Hollestelle et al. concluded conclusively that MDA-MB-435 is a breast cancer cell line [START_REF] Hollestelle | Comment Re: MDA-MB-435 and M14 cell lines: identical but not M14 Melanoma?[END_REF].

We provide strong evidence that intraperitoneally injected N-TSP2-Fc sufficiently reached the blood circulation as shown by an inhibition-ELISA and thereby systemically inhibited MDA-MB-435 tumor progression due to an inhibition of angiogenesis. We could also demonstrate that IgG-Fc did not have any major effects on MDA-MB-435 tumor growth in vivo.

In addition, the inhibitory effect of recombinant N-TSP2-Fc on the growth of breast carcinomas could be confirmed by using of another, MDA-MB-231 [START_REF] Cailleau | Breast tumor cell lines from pleural effusions[END_REF], breast cancer cell line. Systemic treatment with N-TSP2-Fc also resulted in a significant inhibition of xenotransplanted MDA-MB-231 breast cancer cells. Our findings are consistent with recently reported growth inhibition of xenotransplanted A431 squamous cell carcinomas by intraperitoneal application of a recombinant TSP-2 protein that include TSR sequences [START_REF] Noh | An N-terminal 80 kDa recombinant fragment of human thrombospondin-2 inhibits vascular endothelial growth factor induced endothelial cell migration in vitro and tumor growth and angiogenesis in vivo[END_REF]. We have combined the accuracy and sensitivity of real-time PCR [START_REF] Mocellin | Quantitative real-time PCR: a powerful ally in cancer research[END_REF] with the human specificity of alu sequences [START_REF] Schneider | Quantification of human Alu sequences by real-time PCR--an improved method to measure therapeutic efficacy of anti-metastatic drugs in human xenotransplants[END_REF][START_REF] Van Der Horst | TaqManbased quantification of invasive cells in the chick embryo metastasis assay[END_REF]54] for direct quantitative evaluation of metastatic MDA-MB-435 breast cancer cells in lymph nodes and lungs. As early as two weeks after tumor cell injection we could detect human tumor cell DNA in lymph node and lung tissue of MDA-M-435 breast tumor bearing mice. The amount of human DNA in the lungs was much lower than that in the lymph nodes, indicating that lymph node metastasis is an earlier event than distant lung metastasis. This is consistent with the clinical course of most breast cancer diseases [START_REF] Muller | Current status in human breast cancer micrometastasis[END_REF][START_REF] Nakagawa | Detection of circulating tumor cells in early-stage breast cancer metastasis to axillary lymph nodes[END_REF]. We also found significantly less human tumor cell DNA in the lymph nodes and lungs of N-TSP2-Fc treated mice as compared to control treated mice. The inhibitory effect on metastasis was maintained over the entire period of in vivo tumor growth. Even at five weeks after tumor cell inoculation, both lymph node and lung metastasis were significantly reduced in N-TSP2-Fc treated mice.

Controversial findings have been reported regarding the effects of TSP-1 on metastasis [START_REF] Albo | Thrombospondin-1 and transforming growth factor-beta l promote breast tumor cell invasion through up-regulation of the plasminogen/plasmin system[END_REF][START_REF] Albo | Tumour cell thrombospondin-1 regulates tumour cell adhesion and invasion through the urokinase plasminogen activator receptor[END_REF] [47] [START_REF] Weinstat-Saslow | Transfection of thrombospondin 1 complementary DNA into a human breast carcinoma cell line reduces primary tumor growth, metastatic potential, and angiogenesis[END_REF]. Recent in vitro [START_REF] Albo | Thrombospondin-1 and transforming growth factor-beta l promote breast tumor cell invasion through up-regulation of the plasminogen/plasmin system[END_REF][START_REF] Arnoletti | Thrombospondin and transforming growth factor-beta 1 increase expression of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 in human MDA-MB-231 breast cancer cells[END_REF] and in vivo [START_REF] Yee | The effect of thrombospondin-1 on breast cancer metastasis[END_REF] data suggest that prometastatic effects of endogenous TSP-1 may involve the activation of transforming growth factor-ß (TGFß) via the unique sequence KRFK found between the first and second type 1 repeats of TSP-1 [START_REF] Schultz-Cherry | Regulation of transforming growth factor-beta activation by discrete sequences of thrombospondin 1[END_REF]. Noteworthy, TSP-2 lacks the KRFK sequence and recent studies have not found any evidence for activation of latent TGF-ß by TSP-2 [START_REF] Kyriakides | Altered extracellular matrix remodeling and angiogenesis in sponge granulomas of thrombospondin 2-null mice[END_REF]. Whether the lack of TGF-ß activation may explain the lack of any reported prometastatic effect of TSP-2 remains to be further evaluated. In support of this hypothesis are recent findings demonstrating that lymph node metastasis was only observed in TSP-2-null mice but not in wild-type mice in a multistep chemically-induced skin carcinogenesis protocol [START_REF] Hawighorst | Thrombospondin-2 plays a protective role in multistep carcinogenesis: a novel host anti-tumor defense mechanism[END_REF] whereas in a transgenic mouse model of breast cancer pulmonary metastasis was increased in TSP-1 null mice as compared to wildtype mice [START_REF] Yee | The effect of thrombospondin-1 on breast cancer metastasis[END_REF]. Recently, preclinical studies reported that angiogenesis inhibitors targeting the VEGF signaling pathway reduced primary tumor growth but promoted tumor invasiveness and metastasis in vivo [START_REF] Ebos | Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis[END_REF][START_REF] Paez-Ribes | Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis[END_REF]. Multi-targeting of parallel angiogenesis regulating pathways have been proposed to improve the benefits of antiangiogenic drug therapy [START_REF] Bergers | Modes of resistance to anti-angiogenic therapy[END_REF]. Our results demonstrate that N-TSP2-Fc treatment potently inhibits both tumor growth and metastasis in vivo mediated by CD36-dependent inhibition of tumor angiogenesis and they indicate its potential use for antiangiogenic combination therapies to treat breast cancer.

Figure legends

Tables Table 1. Intraperitoneal injection of N-TSP2-Fc results in increased serum level of TSP-2.

After intraperitoneal injection of 100 µg of N-TSP2-Fc diluted in 100 µl PBS serum levels of TSP-2 was measured by an inhibition-ELISA using an antibody directed against the N-terminal region of TSP2. Data revealed that serum concentration of TSP-2 rapidly increased (0.5 h) and reached a peak at 1 h after intraperitoneal injection. The serum concentration of TSP-2 was increased for 24 h.

Fig. 1 .

 1 Fig. 1. N-TSP2-Fc inhibits HDMEC tube formation on Matrigel in a CD36-dependent manner. HDMEC were seeded on Matrigel in the presence of VEGF. Cultures were treated

Fig. 2 .

 2 Fig. 2. N-TSP2-Fc induces apoptosis of HDMEC by CD36 receptor mediated loss of mitochondrial membrane potential and activation of caspase-3. Percentage of apoptotic

Fig. 3 .

 3 Fig. 3. Systemic treatment with N-TSP2-Fc inhibits in vivo growth and angiogenesis of MDA-MB-435 tumors grown in the mammary gland. PBS (control) or 5 mg per kg body

Fig. 4 .

 4 Fig. 4. N-TSP2-Fc inhibits lymph node and lung metastasis. Quantitative real-time PCR

Fig. 1S .

 1S Fig. 1S. Systemic application of IgG-Fc does not inhibit in vivo growth of MDA-MB-435

Fig. 2S .

 2S Fig. 2S. Systemic treatment with N-TSP2-Fc inhibits in vivo growth of MDA-MB-435

Acknowledgments

We thank Renate Dietrich, Matthias Läsche, and Hiltrud Schulz for excellent technical assistance. This work was supported by Deutsche Forschungsgemeinschaft grants (HA 2898 4-1 and 4-2, MK SFB 832 A7).