Sylvie Rodrigues-Ferreira

Anne Nehlig

Clarisse Monchecourt

Sarah Nasr

Laetitia Fuhrmann

Magali Lacroix-Triki

Ingrid Garberis

Véronique Scott

Suzette Delaloge

Barbara Pistilli

Magali Lacroix- Triki

Philippe Vielh

Thierry Dubois

Anne Vincent-Salomon

Fabrice Andre

Clara Nahmias
email: clara.nahmias@inserm.fr

Combinatorial expression of microtubule-associated EB1 and ATIP3 biomarkers improves breast cancer prognosis

Keywords: MAPRE1, MTUS1, diagnosis, prognosis, breast cancer, biomarkers combination

Introduction

Breast cancer is a complex disease whose clinical management relies on well-established clinico-pathological characteristics and molecular biomarkers. The emergence of highthroughput techniques for molecular profiling of breast tumors, such as DNA arrays and RNAseq, has allowed extensive progress in the diagnosis, classification and prognosis of breast tumors. The availability of large molecular databases now provides the opportunity to rapidly analyze individual and combinatorial expression of genes in cohorts of breast cancer patients, which may accelerate the identification of novel molecular biomarkers that are urgently needed for better stratifying breast cancer patients and deciding the type of therapy to be administered.

The microtubule (MT) cytoskeleton plays a key role in various biological processes such as intracellular transport, cell migration and mitosis, all of which are altered in cancer. MTs are polarized protofilaments that constantly alternate between phases of polymerization (growth) and depolymerization (shrinkage) at their plus ends [START_REF] Mitchison | Dynamic instability of microtubule growth[END_REF][START_REF] Howard | Dynamics and mechanics of the microtubule plus end[END_REF]. This dynamic behavior is essential for MTs to explore the cytosol and ensure cell homeostasis, and is tightly regulated by a wide number of MT-associated proteins (MAPs) [START_REF] Akhmanova | Tracking the ends: a dynamic protein network controls the fate of microtubule tips[END_REF]. Any defect in these regulatory proteins may alter the organisation and/or function of MTs, with major consequences on cancer initiation or progression.

End-binding protein 1 (EB1) is the leader member of a subfamily of MAPs including EB2 and EB3, encoded by homologous genes designated MAPRE1, MAPRE2 and MAPRE3, respectively [START_REF] Su | Characterization of human MAPRE genes and their proteins[END_REF]. EB1 and EB3 have been extensively studied, EB1 being ubiquitous and EB3 being predominant in the brain, whereas EB2 remains less characterized [START_REF] Nehlig | Regulation of end-binding protein EB1 in the control of microtubule dynamics[END_REF]. EB1 preferentially binds and accumulates at growing MT ends and is considered a surrogate marker of MT dynamics [START_REF] Vaughan | TIP maker and TIP marker; EB1 as a master controller of microtubule plus ends[END_REF]. Upon binding to MT ends, EB1 accelerates the rate of catastrophe (rapid depolymerization) and thus also functionnally contributes to MT-end maturation and dynamic instability [START_REF] Maurer | EB1 accelerates two confirmational transitions important for microtubule maturation and dynamics[END_REF]. In addition, EB1 recruits a large number of regulatory proteins at MT plus ends, thereby orchestrating the regulation of MT dynamics and MT targeting to organelles [START_REF] Galjart | Plus-end-tracking proteins and their interactions at microtubule ends[END_REF][START_REF] Jiang | A proteome-wide screen for Mammalian SxIP Motif-containing microtubule plus-end tracking proteins[END_REF][START_REF] Kumar | +TIPs: SxIPping along microtubule ends[END_REF].

The pivotal role of EB1 on MT dynamics suggests that alterations of EB1 expression or function may have important consequences in cancer. Indeed, up-regulation of EB1 protein in tumor samples has been reported in breast cancer [START_REF] Dong | Oncogenic function of microtubule end-binding protein 1 in breast cancer[END_REF], glioblastoma [START_REF] Berges | End-binding 1 protein overexpression correlates with glioblastoma progression and sensitizes to Vinca-alkaloids in vitro and in vivo[END_REF], hepatocarcinoma [START_REF] Orimo | Proteomic profiling reveals the prognostic value of adenomatous polyposis coli-end-binding protein 1 in hepatocellular carcinoma[END_REF], oral [START_REF] Kumar | End Binding 1 (EB1) overexpression in oral lesions and cancer : A biomarker of tumor progression and poor prognosis[END_REF] and colorectal cancer [START_REF] Stypula-Cyrus | End-binding protein 1 (EB1) up-regulation is an early event in colorectal carcinogenesis[END_REF][START_REF] Sugihara | Proteomic-based identification of the APC-binding protein EB1 as a candidate of novel tissue biomarker and therapeutic target for colorectal cancer[END_REF]. In breast cancer, high EB1 protein levels have been shown to correlate with tumor malignancy and high tumor grade [START_REF] Dong | Oncogenic function of microtubule end-binding protein 1 in breast cancer[END_REF]. However, the status of MAPRE1 gene encoding EB1, and its paralogs MAPRE2 and MAPRE3, has not yet been evaluated in breast cancer, and it remains to be established whether EB1 represents a prognostic biomarker of breast cancer patient survival.

Recent studies have identified several structural MAPs as endogenous antagonists of EB1 functions at MT plus ends [START_REF] Nehlig | Regulation of end-binding protein EB1 in the control of microtubule dynamics[END_REF]. Among them, the ATIP3 protein encoded by candidate tumor suppressor gene MTUS1 has been described as a prognostic biomarker down-regulated in invasive breast cancer [START_REF] Rodrigues-Ferreira | 8p22 MTUS1 gene product ATIP3 is a novel anti-mitotic protein underexpressed in invasive breast carcinoma of poor prognosis[END_REF][START_REF] Molina | ATIP3, a novel prognostic marker of breast cancer patient survival, limits cancer cell migration and slows metastatic progression by regulating microtubule dynamics[END_REF]. ATIP3 is a potent MT-stabilizer that markedly reduces breast tumor growth and distant metastasis [START_REF] Molina | ATIP3, a novel prognostic marker of breast cancer patient survival, limits cancer cell migration and slows metastatic progression by regulating microtubule dynamics[END_REF]. ATIP3 directly binds to EB1 in the cytosol and prevents its turnover and accumulation at growing MT ends, thereby reducing MT dynamics and cell polarity [START_REF] Velot | Negative regulation of EB1 turnover at microtubule plus ends by interaction with microtubule-associated protein ATIP3[END_REF]. These data suggest that altered expression of either ATIP3 or EB1 in breast tumors may impact the levels of intracellular ATIP3-EB1 molecular complexes that govern EB1 function at MT plus ends. This raises the interesting possibility that deleterious effects of high EB1 levels in breast tumors may be moderated by high levels of ATIP3 whereas tumors with high-EB1 and low-ATIP3 levels may remain more aggressive.

The present study investigates the value of MAPRE genes, alone or in combination with MTUS1, for molecular classification, diagnosis and prognosis of breast cancer patients. We show that high MAPRE1but not MAPRE2 and MAPRE3 -levels correlate with tumor malignancy and poor clinical outcome for breast cancer patients, and that combinatorial analysis of MAPRE1 and MTUS1 expression refines breast cancer diagnosis and prognosis compared to MAPRE1 alone.

Methods

Breast tumor samples and gene arrays

In a first cohort of patients (Cohort#1) designed for tumor classification, samples were obtained by fine-needle aspiration of breast lesions from patients referred to the breast diagnosis center of the Gustave Roussy Center (Villejuif, France) between 2006 and 2007 [START_REF] André | Exonic expression profiling of breast cancer and benign lesions: a retrospective analysis[END_REF]. 165 samples, among which 45 benign and 120 malignant tumors, were profiled on a Splice Array (Exonhit, France) [START_REF] Fehlbaum | A microarray configuration to quantify expression levels and relative abundance of splice variants[END_REF]. Patient characteristics and probeset intensities are presented in Supplemental Table S1.

The exploratory cohort of breast cancer patients (Cohort#2), designed for prognostic purposes, comprises 150 infiltrating breast carcinomas obtained from patients who were included between 1988 and 1999 in the prospective database initiated in 1981 by the Institut Curie Breast cancer group (Curie, Paris, France) [START_REF] Rodrigues-Ferreira | 8p22 MTUS1 gene product ATIP3 is a novel anti-mitotic protein underexpressed in invasive breast carcinoma of poor prognosis[END_REF]. Samples were analyzed by Affymetrix HG-U133 DNA array hybridization as described [START_REF] Rodrigues-Ferreira | 8p22 MTUS1 gene product ATIP3 is a novel anti-mitotic protein underexpressed in invasive breast carcinoma of poor prognosis[END_REF][START_REF] Molina | ATIP3, a novel prognostic marker of breast cancer patient survival, limits cancer cell migration and slows metastatic progression by regulating microtubule dynamics[END_REF][START_REF] Reyal | Visualizing chromosomes as transcriptome correlation maps: evidence of chromosomal domains containing co-expressed genes-a study of 130 invasive ductal breast carcinomas[END_REF]. Patients included in the study were aged 33 to 88 and were treated by radiotherapy combined or not with hormono-or chemotherapy after chirurgical resection of the tumor. Immunohistological levels of estrogen receptor (ER) and progesterone receptor (PR) were recorded according to standardized guidelines using 10% as the cut-off for ER and PR positive cells [START_REF] Hammond | American society of clinical oncology/college of american pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer[END_REF]. For human epidermal growth factor receptor-2 (HER2), only membrane staining was considered with a 30% cut-off as recommended [START_REF] Wolff | Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Focused Update[END_REF]. Patient characteristics and probeset intensities are presented in Supplemental Table S2. Two independent series of breast cancer patients, designated Cohort#3 [START_REF] Maire | Polo-like kinase 1: a potential therapeutic option in combination with conventional chemotherapy for the management of patients with triple-negative breast cancer[END_REF][START_REF] Maire | TTK/hMPS1 is an attractive therapeutic target for triple-negative breast cancer[END_REF] and Cohort#4 [START_REF] Molina | ATIP3, a novel prognostic marker of breast cancer patient survival, limits cancer cell migration and slows metastatic progression by regulating microtubule dynamics[END_REF] are described in Supplemental Methods and Supplemental Tables S3 andS4.

Immunohistochemical analysis of breast cancer tissue microarrray (TMA)

Samples of invasive ductal carcinomas (IDCs) were surgically removed before any radiation, hormonal or chemotherapy treatment at Institut Curie from 2005 to 2006 [START_REF] Lodillinsky | p63/MT1-MMP axis is required for in situ to invasive transition in basal-like breast cancer[END_REF]. Patient characteristics and clinical data are presented in Supplemental Table S5. TMA consisted of replicate 1-mm diameter tumor cores selected from whole-tumor tissue section in two most representative tumor areas (high tumor cell density) of each tumor sample. Alcohol formalin acetic acid-fixed paraffin-embedded samples were analyzed by immunohistochemistry (IHC) staining using EnVision FLEX kit (Dako) according to the manufacturer's instructions. For EB1 staining, heat-mediated antigen retrieval was performed in Ethylene-Diamine-Tetra-Acetic acid (EDTA) buffer pH 6 in water bath for 30 min and monoclonal mouse anti-EB1 antibodies (BD Biosciences) diluted 1:300 were incubated overnight at 4°C. ATIP3 staining was performed as previously described [START_REF] Rodrigues-Ferreira | 8p22 MTUS1 gene product ATIP3 is a novel anti-mitotic protein underexpressed in invasive breast carcinoma of poor prognosis[END_REF] using monoclonal anti-MTUS1 antibodies (Abnova) diluted 1 :100. Slides were counterstained with hematoxylin. HeLa cells transfected with control siRNA were used as positive control for EB1 staining, and cells transfected for 48h with EB1-specific siRNA (Dharmacon) [START_REF] Velot | Negative regulation of EB1 turnover at microtubule plus ends by interaction with microtubule-associated protein ATIP3[END_REF] were considered as negative control. Silencing efficiency was validated by immunoblotting using anti-EB1 antibodies (BD Biosciences) diluted 1 :1000 and anti-alpha-tubulin antibodies (Sigma) diluted 1 :1000 as internal control.

EB1 immunoreactivity in tissue sections was classified semi-quantitatively into 5 classes (0, <1, 1, 2, 3) according to the intensity of cytoplasmic staining of tumor cells. Scores for EB1 immunostaining were defined as 0 (undetectable staining on high-power field (x40), <1 (only visible on high-power field (x40), 1 (detected on medium-power field (x10-x20) but well visualized on high-power field (x40), 2 (detected on low-power field (x4-x5) but well visualized on medium-power field (x20), and 3 (well visualized on low-power field (x4-x5). Samples were independently evaluated by two pathologists of different institutes. EB1 expression was classified on a semi-quantitative basis, immunostaining was considered weak for a score from 0 to 1 and strong for a score of 2 to 3. Scoring for ATIP3 staining was evaluated as described [START_REF] Rodrigues-Ferreira | 8p22 MTUS1 gene product ATIP3 is a novel anti-mitotic protein underexpressed in invasive breast carcinoma of poor prognosis[END_REF].

Statistical analysis

Statistical analyses were done using JMP-7 and GraphPad Prism 6.0 softwares. The association between clinicopathological characteristics and the expression level of either MAPRE1, MTUS1, or their combination were calculated using the Mann-Whitney test for continuous variables and using the chi-squared and the Fisher exact tests for dichotomized variables. Dot plot analyses were done using Mann-Whitney test. Overall survival (OS) and Relapse-free survival (RFS) curves were plotted according to the method of Kaplan-Meier and compared by the log-rank test. P < 0.05 was considered statistically significant.

Results

Combinatorial expression of MAPRE1 and MTUS1 genes improves breast cancer diagnosis

Expression levels of the MAPRE1 gene encoding End-Binding protein EB1 were evaluated in a splice-array profiling of 165 breast samples including 120 malignant cancers and 45 benign lesions (Cohort#1) [START_REF] André | Exonic expression profiling of breast cancer and benign lesions: a retrospective analysis[END_REF]. Only T probesets -that identify expressed exons -were taken into account to avoid potential bias due to alternative exon splicing. Analysis of probesets intensities revealed that MAPRE1 transcripts are significantly higher in malignant compared to benign breast tumors (Fig. 1a and Supplemental Fig. S1). Heatmap analysis of MAPRE1 gene expression was then used to classify tumor samples according to high or low levels of EB1 transcripts (Fig. 1b). As shown in Fig. 1c and Table 1, 65% of low-MAPRE1 expressing tumors were malignant compared to 79,6% of those expressing high-MAPRE1 levels, further indicating that elevated expression of MAPRE1 gene is associated with tumor malignancy. In contrast, expression levels of paralog MAPRE2 and MAPRE3 genes encoding EB2 and EB3 proteins, respectively, were either decreased (Supplemental Fig. S2a) or unchanged (Supplemental Fig. S2b) in cancer samples compared to benign lesions.

Expression of the MTUS1 gene, whose major product ATIP3 antagonizes EB1 functions [START_REF] Nehlig | Regulation of end-binding protein EB1 in the control of microtubule dynamics[END_REF][START_REF] Velot | Negative regulation of EB1 turnover at microtubule plus ends by interaction with microtubule-associated protein ATIP3[END_REF], was analyzed in the same samples (Supplemental Table S1). Analysis of MTUS1 probesets intensities (Fig. 1d and Supplemental Fig. S3) revealed that MTUS1 levels are significantly decreased in malignant breast tumors compared to benign lesions. Classification of samples into clusters expressing high-and low-MTUS1 using heatmap analysis (Fig. 1e) further indicated that 93,6% of low-MTUS1 tumors were malignant compared to 54% of tumors with high-MTUS1 levels (Fig. 1f, Table 1).

We next evaluated the diagnostic value of combining MAPRE1 and MTUS1 gene expression.

Tumors expressing high-MAPRE1 and low-MTUS1 levels included a majority (97.7%) of malignant samples, compared to 45.2% among those expressing low-MAPRE1 and high-MTUS1 (Fig. 1g). Of interest, combining MAPRE1 and MTUS1 gene expression allowed better tumor classification compared to each biomarker alone (Fig. 1g, Table 1). Together these studies point to MAPRE1 and MTUS1 as valuable diagnostic biomarkers and suggest that analyzing combinatorial expression of these genes may refine tumor classification and improve breast cancer diagnosis.

Combinatorial expression of MAPRE1 and MTUS1 genes correlates with tumor grade

Analysis of MAPRE1 gene levels in invasive breast carcinomas from 150 patients of the Curie Institute (Exploratory Cohort#2) revealed that MAPRE1 expression is significantly increased in high grade (GIII) tumors compared to those of histological grade I (p= 0.0003) and grade II (p=0.0242) (Fig. 2a, Table 2). Tumors were classified into clusters expressing low or high levels of EB1 transcripts according to heat-map analysis of MAPRE1 probesets intensities (Fig. 2b).

Results indicate that the percentage of grade-III tumors is 3 times more elevated among high-MAPRE1 (36,8%) compared to low-MAPRE1 (10,7%) expressing tumors (Fig. 2c, Table 2).

Conversely, grade-I tumors were significantly less abundant among high-MAPRE1 (18,4%) compared to low-MAPRE1 (49,1%) groups (Table 2), indicating that high transcript levels correlate with high histological grade.

Two independent series of 130 and 155 invasive carcinoma from breast cancer patients of the Curie Institute (Cohort#3) (Supplemental Table S3) and Gustave Roussy Hospital (Cohort #4) (Supplemental Table S4) were then analyzed. As shown in Supplemental Fig. S4a, MAPRE1 gene levels were higher in cancer samples compared to 11 normal tissues. In both sets of tumors, MAPRE1 levels were increased in grade-III versus grade-I/II invasive tumors (Supplemental Fig. S4a,S4d). Acordingly, the percentage of high-grade tumors was also elevated in high-MAPRE1 compared to low-MAPRE1 breast tumors from Cohorts #3 and #4 (Supplemental Fig. S4b, S4c, S4e, S4f and Supplemental Table S6, S7), therefore confirming that high-MAPRE1 correlates with tumor aggressiveness. Of note, MAPRE1 levels were not significantly different among ER-positive and ER-negative breast tumors of Cohort#2 (Supplemental Fig. S4g) and Cohort#4 (Supplemental Fig. S4h), although in Cohort#3, high MAPRE1 levels were significantly associated with ER-negative tumors (Suppl Fig. S4i and Supplemental Table S6). Of note, the percentage of ER-negative tumors included in Cohort #2 and #4 is very low (18,7% and 6,6%, respectively) compared to that in Cohort#3 (54,6%), which may explain why results do not reach significance in these two series of patients. Future studies including new cohorts with larger numbers of ER-negative breast cancer samples are warranted to address that question.

We then investigated whether MAPRE2 and MAPRE3 genes may also be regulated in invasive breast cancer. Probesets intensities for these two genes were examined in each cohort of invasive breast carcinomas when data were available, and results (Supplemental Fig. S5) revealed that MAPRE2 and MAPRE3 gene levels do not consistently correlate with tumor grade.

Together, these studies indicate that increased levels of MAPRE1, but not MAPRE2 or MAPRE3 genes, correlate with high tumor grade in breast cancer.

Previous studies have shown that MTUS1 gene is down-regulated in breast cancer samples compared with normal tissue, and that low-MTUS1 levels correlate with high tumor grade [START_REF] Rodrigues-Ferreira | 8p22 MTUS1 gene product ATIP3 is a novel anti-mitotic protein underexpressed in invasive breast carcinoma of poor prognosis[END_REF][START_REF] Molina | ATIP3, a novel prognostic marker of breast cancer patient survival, limits cancer cell migration and slows metastatic progression by regulating microtubule dynamics[END_REF]. We thus investigated the impact of combinatorial MAPRE1 and MTUS1 expression on histological grade. Based on heatmap classification of 150 tumors of Cohort#2 according to MAPRE1 and MTUS1 probesets intensities, four clusters of tumors expressing either low or high levels of each transcript were established (Fig. 2d). Comparison with histological grade revealed that cluster 3 -expressing high-MAPRE1 and low-MTUS1 levels -is significantly associated with higher percentage of high-grade tumors (44.4% grade-III tumors in cluster 3 compared to 18.3%, 2% and 18.2% in clusters 1, 2 and 4, respectively) (Fig. 2e, Table 2). Consistently, 46.1% of grade-III tumors were from cluster 3, compared to 18% and 6.4% of grade-II and grade-I tumors, respectively (Fig. 2f). Interestingly, among high-MAPRE1 expressing tumors that are the most aggressive, those with low-MTUS1 (cluster 3) were predominantly of high grade compared to those with high-MTUS1 (cluster 4) (Fig. 2e), indicating that combined expression of MAPRE1 and MTUS1 genes better distinguishes tumor grade than MAPRE1 alone. Similar results were obtained using two independent sets of tumors (Supplemental Tables S6 andS7, Supplemental Fig. S6). Of note, aggressive tumors from cluster 3 represent a substantial proportion of all breast tumors (18% in Cohort#2, 31.5% in Cohort#3 and 23.9% in Cohort#4) (Table 2 and Supplemental Tables S6 andS7).

Combinatorial expression of MAPRE1 and MTUS1 improves breast cancer prognosis

The prognostic value of MAPRE1 in breast cancer was assessed by comparing probesets intensities with clinical data of patient survival. The probability of overall survival was significantly reduced in patients with breast tumors expressing high MAPRE1 compared with low MAPRE1 levels (HR 2.22; 95% CI 1.19-4.12; p=0.0058) (Fig. 3a and Table 3). The Kaplan-Meier plotter tool available online was also used to interrogate public databases [START_REF] Gyorffy | An online survival analysis tool to rapidly assess the effect of 22,277 genes on breast cancer prognosis using microarray data of 1809 patients[END_REF]. High MAPRE1 expression correlated with poorer overall survival in 1402 breast cancer patients (Supplemental Fig. S7a) and poorer relapse-free survival in 3115 patients (Supplemental Fig. S7b), further comforting our results that high-MAPRE1 levels are associated with poor prognosis in breast cancer.

To further investigate whether combinatorial expression of MAPRE1 and MTUS1 may have improved prognostic value compared to each single gene, we evaluated the probability of survival among tumor clusters classified according to MAPRE1 and MTUS1 probeset intensities as defined previously (Fig. 2d). Results indicate that overall survival and relapse-free survival are significantly reduced (p=0.0001) for patients of cluster 3 -expressing high-MAPRE1 and low-MTUS1 levels -compared to all other clusters (Fig. 3b,3c, Table 2 and Supplemental Fig. S7c-d, Table S6). Accordingly, the percentage of patients surviving after 5 years was lower for tumors of cluster 3 (42%) compared to those of cluster 1 (61%), cluster 2 (85%) and cluster 4 (60%) (Fig. 3d). Thus, combining MAPRE1 and MTUS1 gene levels improves breast cancer prognosis compared to MAPRE1 or MTUS1 alone. Multivariate analyses including ER, PR, HER2, tumor grade, MAPRE1 and MTUS1 gene levels further revealed that MAPRE1/MTUS1 gene combination (HR 3.50; CI 1.12-10.85; p 0.015) is an independent indicator of overall survival whereas MAPRE1 alone (HR 1.40; CI 0.40-4.89; p 0.299) is not (Table 3). Notably, MTUS1 (HR 2.7; CI 1.34-5.42; p 0.002) is also identified as an independent prognostic factor, in line with previous studies [START_REF] Molina | ATIP3, a novel prognostic marker of breast cancer patient survival, limits cancer cell migration and slows metastatic progression by regulating microtubule dynamics[END_REF].

We then turned to IHC experiments to evaluate whether combined expression of EB1 and ATIP3 at the protein level may also improve breast cancer prognosis. Experimental conditions set up for EB1 staining (Supplemental Fig. S7e) were used to detect EB1 expression in patient biopsies. As expected, EB1 immunostaining was weak in normal breast and was markedly increased in the cytosol of tumor samples (Supplemental Fig. S7f). Tissue microarrays from 212 invasive breast tumors were then analyzed (Supplemental Table S5) and EB1 immunostaining (Fig. 3e) was compared with data for patients survival. Results show that the probability of overall survival was reduced in patients with tumors showing strong EB1 staining (HR 3.46; CI 1.065-7.284; p=0.037) (Fig. 3f,Table 4), therefore validating the EB1 protein as a prognostic biomarker in breast cancer.

To further evaluate the impact of EB1-ATIP3 combination on patient survival, serial sections of the TMA were stained with anti-ATIP antibodies and samples were classified according to strong or weak EB1 and ATIP3 immunostaining (Fig. 3g and Supplemental Table S5). Results indicate that the probability of survival is significantly reduced in patients with tumors expressing strong-EB1 and weak-ATIP3 compared to all other groups (p=0.0153) (Fig. 3h), which comforts the data obtained at the mRNA level (Fig. 3b). Consistent with previous results on DNA array analyses, this group of aggressive breast tumors represents 30.4% of all tumor samples (Table 4). Together, these data confirm DNA array results and demonstrate the improved prognostic value of combinatorial EB1/ATIP3 expression in breast cancer.

Discussion

This study examined the diagnostic and prognostic value of MAPRE genes expression in breast cancer, either alone or in combination with candidate tumor suppressor MTUS1 gene. We found that MAPRE1, but not MAPRE2 nor MAPRE3, is up-regulated in malignant tumors compared to benign lesions, and in high-grade compared to low-grade invasive carcinoma. These results are consistent with previous studies [START_REF] Kumar | +TIPs: SxIPping along microtubule ends[END_REF] reporting EB1 as an oncogenic protein up-regulated in malignant and high-grade breast tumors. In contrast to this latter study however, our data did not clearly correlate EB1 levels with ER status in breast tumors and more studies are required to investigate this question. Results presented here further indicate that high expression of MAPRE1 gene and encoded protein EB1 correlates with reduced survival of the patients, pointing out this molecule as a novel prognostic biomarker in breast cancer.

We further demonstrate here that combinatorial expression of MAPRE1 and MTUS1 genes significantly improves breast cancer diagnosis and prognosis compared to MAPRE1 and MTUS1 alone. Combining data on MAPRE1 and MTUS1 expression both at the gene and protein levels allowed to identify a group of aggressive high-grade tumors with high-EB1 and low-ATIP3 levels, that represent 20% to 30% of all breast tumors and are associated with reduced patient survival rates. While a number of studies have identified gene signatures associated with poor prognosis of breast cancer patients [START_REF] Alexe | Breast cancer prognosis by combinatorial analysis od gene expression data[END_REF][START_REF] Espinosa | Gene profiling in breast cancer: time to move forward[END_REF], this is to our knowledge the first demonstration that combinatorial expression of two defined biomarkers with known associated molecular mechanisms may be used as a tool to select populations of cancer patients for personalized therapy. Further studies are warranted to evaluate the feasibility of using MAPRE1 and MTUS1 biomarkers as diagnostic and prognostic tools in the clinic.

On a molecular basis, these results are supported by our previous findings that ATIP3 acts as a brake on EB1 functions through binding and reducing EB1 turnover at MT growing ends [START_REF] Velot | Negative regulation of EB1 turnover at microtubule plus ends by interaction with microtubule-associated protein ATIP3[END_REF].

In the absence of ATIP3, EB1 accumulates at MT ends and forms large comet-like structures that specify increased MT dynamics [START_REF] Molina | ATIP3, a novel prognostic marker of breast cancer patient survival, limits cancer cell migration and slows metastatic progression by regulating microtubule dynamics[END_REF]. Results on breast cancer patients favor a model in which in breast tumors with low ATIP3 levels, the brake is turned off and the oncogenic activity of EB1 is increased. This opens the way to novel molecular therapeutic strategies involving delivery of active domains of ATIP3 to target the population of high-EB1/low-ATIP3 breast tumors that remain of poor prognosis. Alternative approaches may rely on the design or discovery of small molecule modulators of EB1 binding at MT ends to compensate for ATIP3 loss in the target population of breast tumors identified here. An integrative approach based on the discovery of small molecule scaffolds that target EB1 interactions with other partners at MT ends has indeed been recently described [START_REF] Almeida | Targeting SxIP-EB1 interaction: An integrated approach to the discovery of small molecule modulators of dynamic binding sites[END_REF].

In conclusion, we show here for the first time that combinatorial expression of MT-associated EB1 and ATIP3 biomarkers improves breast cancer prognosis compared to each biomarker alone, and may be useful for better stratifying the patients for targeted molecular therapy.

Further studies are warranted to analyze combinations of MAPRE1 and MTUS1 genes expression in other types of cancers such as glioblastoma [START_REF] Dong | Oncogenic function of microtubule end-binding protein 1 in breast cancer[END_REF], hepatocarcinoma [START_REF] Berges | End-binding 1 protein overexpression correlates with glioblastoma progression and sensitizes to Vinca-alkaloids in vitro and in vivo[END_REF], and oral [START_REF] Orimo | Proteomic profiling reveals the prognostic value of adenomatous polyposis coli-end-binding protein 1 in hepatocellular carcinoma[END_REF] and colorectal [START_REF] Stypula-Cyrus | End-binding protein 1 (EB1) up-regulation is an early event in colorectal carcinogenesis[END_REF] cancers, in which high-EB1 protein levels have been shown to correlate with poor clinical outcome.

Legends to Figures

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Figure 1 :

 1 Figure 1: Combinatorial expression of MAPRE1 and MTUS1 genes improves breast cancer diagnosis

Figure 2 :

 2 Figure 2: Combinatorial expression of MAPRE1 and MTUS1 genes correlates with tumor grade

Figure 3 :

 3 Figure 3: Combinatorial expression of MAPRE1 and MTUS1 genes improves breast cancer prognosis

Figure 1

 1 Figure 1

Figure 2

 2 Figure 2

Figure 3

 3 Figure 3

Table 1 . Characteristics of patients from Cohort#1, with high or low MAPRE1, MTUS1 or combinatorial genes expression

 1 Tables Low and high MAPRE1 and MTUS1 levels were determined according to heatmap analysis of Exonhit probesets intensities. Tumors of Cluster 3 expressing high-MAPRE1 and low-MTUS1 were compared to tumors of all other clusters.

Table 2 . Characteristics of patients from Cohort #2, with high or low MAPRE1 or MAPRE1/MTUS1 combinatorial genes expression

 2 Low and high MAPRE1 levels were determined according to heatmap analysis of Affymetrix probesets intensities. Tumors of Cluster 3 expressing high-MAPRE1 and low-MTUS1 were compared to tumors of all other clusters.

Table 3 . Univariate and multivariate analysis of the correlation between clinical parameters, MAPRE1 and MTUS1 levels, and survival time of patients from Cohort#2

 3 A star indicates multivariate analysis including MAPRE1/MTUS1 combination. HR: Hazard Ratio; CI: Confidence Interval; p: p-value; p-values that reach significance are in bold.

Table 4 . Characteristics of patients included in Tissue Microarray analysis, with strong or weak expression of EB1 or EB1/ATIP combinations.

 4 Weak and strong EB1 and ATIP3 levels were scored by IHC as indicated in the Methods. Tumors classified as strong-EB1 and weak-ATIP3 were compared to tumors of all other groups.

Table 2 . Characteristics of patients from Cohort#2 with high or low MAPRE1 or combined MAPRE1/MTUS1 genes expression

 2 Low and high MAPRE1 and MTUS1 levels were determined according to heatmap analysis of probesets intensities. Tumors of Cluster 3 expressing high-MAPRE1 and low-MTUS1 were compared to tumors of all other clusters

	Variables
	ER

Table 2

 2 Click here to access/download;Table;SRF Nahmias Table 2.pdf

		HR	95% CI	p	HR	95% CI	p	HR	95% CI	p
	ER -vs +	3.2	1.64-6.2	0.0003	1.89	0.81-4.36	0.068	1.9	0.83-4.3	0.062
	PR -vs +	2.2	1.23-3.9	0.0036	1.5	0.59-3.78	0.195	1.5	0.59-3.78	0.195
	HER2 + vs -	2.8	1.31-5.94	0.0036	1.73	0.67-4.45	0.128	1.67	0.61-4.55	0.158
	Grade III vs I/II	4.1	1.95-8.6	<0.0001	2,00	0.86-4.6	0.051	1.98	0.86-4.57	0.055
	MAPRE1 high vs low	2.22	1.19-4.12	0.0058	1.4	0.40-4.89	0.299
	MTUS1 low vs high	3.07	1.7-5.5	0.0001	2.7	1.34-5.42	0.0018
	MAPRE1 MTUS1 combination cluster 3 vs 2	5.46	2.12-13.97	0.0002				3.5	1.12-10.85	0.015

Table 3 . Univariate and multivariate analysis of the correlation between clinicopathological parameters, MAPRE1 and MTUS1 level, and survival time of patients from Cohort #2

 3 * multivariate analysis including MAPRE1/MTUS1 combination HR: Hazard Ratio, CI: Confidence Interval, p: p-value p-values that reach significance are in bold

	Univariate Analysis	Multivariate Analysis	Multivariate Analysis*

Table 3

 3 Click here to access/download;Table;SRF Nahmias revised Table3.pdf

Acknowledgements

We wish to thank Dr Céline Lefebvre for helpful discussion and Nicolas Signolle and Ibrahim Bouakka for excellent assistance. This work was supported by the INSERM, the CNRS, the University Paris-Saclay, the Institut Gustave Roussy, the A*MIDEX project (n°ANR-11-IDEX-0001-02) funded by the "Investissements d'Avenir" French Government program, managed by the ANR and ITMO Cancer AVIESAN as part of the Cancer Plan No. PC201419, the Labex LERMIT, the Fondation ARC pour la Recherche contre le Cancer, the Ligue Nationale Contre le Cancer, the association Odyssea, GEFLUC and Prolific.

Funding

This study was funded by the A*MIDEX project (n°ANR-11-IDEX-0001-02) funded by the "Investissements d'Avenir" French Government program, managed by the ANR and ITMO Cancer AVIESAN as part of the Cancer Plan No. PC201419. Anne Nehlig was recipient of grants from la Ligue Nationale contre le Cancer, the association Odyssea and Prolific. This study was funded by the Fondation ARC pour la Recherche contre le Cancer, the Labex LERMIT, the association GEFLUC and Prolific.

Compliance with current laws

The authors declare that all experiments presented here comply with the current laws of France

Compliance with ethical standards

Conflict of interests :

The authors declare that they have no conflict of interests.

Ethical approval :

This article does not contain any studies with animals performed by any of the authors.

All procedures used in studies involving human participants have been previously published elsewhere and were in accordance with ethical standards of the Institut Curie and Gustave Roussy committees, and with 1964 Helsinki declaration.

Informed consent was obtained from all individual participants included in ths study.