Sylvie Rodrigues-Ferreira

Angie Molina

PhD Clara Nahmias
email: clara.nahmias@inserm.fr

Microtubule-associated tumor suppressors as prognostic biomarkers in breast cancer

Keywords: ATIP3, Combinatorial expression, EB1, Microtubule-associated protein, Prognosis, Tumor suppressor

Breast cancer is the most common malignancy in women worldwide. Although important therapeutic progress was achieved over the past decade, this disease remains a public health problem. In the light of precision medicine, the identification of new prognostic biomarkers in breast cancer is urgently needed to stratify populations of patients with poor clinical outcome who may benefit from new personalized therapies. The microtubule cytoskeleton plays a pivotal role in essential cellular functions and is an interesting target for cancer therapy. Microtubule assembly and dynamics are regulated by a wide range of microtubule-associated proteins (MAPs), some of which have oncogenic or tumor suppressors effects in breast cancer. This review covers current knowledge on microtubule-associated tumor suppressors (MATS) in breast cancer and their potential value as prognostic biomarkers. We present recent studies showing that combinatorial expression of ATIP3 and EB1, two microtubule-associated biomarkers with tumor suppressor and oncogenic effects, respectively, improves breast cancer prognosis compared to each biomarker alone. These findings are discussed regarding the increasing complexity of protein networks composed of MAPs that coordinate microtubule dynamics and functions. Further studies are warranted to evaluate the prognostic value of combined expression of different MATS and their interacting partners in breast cancer.

Introduction

Breast cancer is a major cause of death in women worldwide. Clinical management of this heterogeneous disease relies on well-established clinico-pathological characteristics and molecular biomarkers, including estrogen and progesterone receptors as well as the HER2 oncogene [START_REF] Harbeck | Breast cancer[END_REF]. The classification of breast tumors into distinct molecular subtypes (Luminal, HER2-positive and triple-negative) is of invaluable help in making clinical decisions. However, tumors from the same subtype still remain heterogeneous in terms of prognosis and response to therapy [START_REF] Falco | Tumour biomarkers: homeostasis as a novel prognostic indicator[END_REF]. Over the past decade, the emergence of high-throughput techniques for molecular profiling and DNA sequencing of breast tumors has allowed extensive progress in the classification, prognosis and treatment of early and advanced breast cancer [START_REF] André | Comparative genomic hybridisation array and DNA sequencing to direct treatment of metastatic breast cancer: a multicentre, prospective trial (SAFIR01/UNICANCER)[END_REF][START_REF] Bertucci | Genomic characterization of metastatic breast cancers[END_REF]. In the light of precision medicine, the continuously increasing accumulation of big data opens new avenues of investigation [START_REF] Low | Breast cancer: The translation of big genomic data to cancer precision medicine[END_REF]. The identification of novel biomarkers enabling personalized therapeutic strategies for selected breast tumors has become a major issue [START_REF] Bettaieb | Precision medicine in breast cancer: reality or utopia?[END_REF]. An important point to reach is the ability to identify prognostic biomarkers to stratify populations of breast cancer patients at high risk to relapse and develop metastasis. Selecting these patients is a prerequisite to the development of targeted therapies.

The microtubule cytoskeleton is an essential cellular component that plays a key role in various biological processes such as cell division, polarity and migration, all of which are altered in cancer.

Microtubules are polarized protofilaments

-tubulin heterodimers that are organized head-to-tail and grow from the minus to the plus end. These are highly dynamic structures, constantly switching between phases of polymerization (growth) and depolymerization (shrinkage) at their plus ends, a process called dynamic instability. Dynamic instability allows the microtubule cytoskeleton to explore the cytosol and rapidly reorganize in response to external cues to ensure appropriate cell function. Coordinated regulation of microtubule dynamics during mitosis and interphase permits mitotic spindle formation and orientation, chromosomes segregation towards cell poles during anaphase, cytoskeleton and cell shape remodeling, intracellular trafficking and signaling as well as cell migration and polarization.

Microtubule assembly and dynamics are tightly regulated by a large number of microtubule-associated proteins (MAPs) that interact with tubulin or polymerized microtubules to either stabilize or destabilize the network. A subset of MAPs, known as kinesins, function as molecular motors to ensure intracellular transport of proteins and organelles. Some MAPs localize along the microtubule lattice (structural MAPs) whereas others preferentially bind to microtubule plus ends (+TIPs) or minus ends (-TIPs) [START_REF] Akhmanova | Control of microtubule organization and dynamics: two ends in the limelight[END_REF].

At the plus end, End-Binding proteins (EB1, EB2, EB3) play a major role as scaffolds to recruit a wide range of plus-end tracking proteins that contribute to the regulation of microtubule dynamics and targeting of organelles [START_REF] Akhmanova | Control of microtubule organization and dynamics: two ends in the limelight[END_REF][START_REF] Jiang | A Proteome-wide screen for mammalian SxIP motif-containing microtubule plus-end tracking proteins[END_REF]. Minus ends are occupied by large complexes of MAPs, also including EB1, that cooperate to regulate microtubule nucleation and dynamics [START_REF] Martin | Coming into Focus: Mechanisms of Microtubule Minus-End Organization[END_REF].

Because they regulate the organization and function of the microtubule cytoskeleton in interphase and mitosis, MAPs can be considered as the "guardians" of cellular integrity. Alterations in the sequence or expression levels of these proteins may lead to cytoskeletal defects with major consequences on cancer initiation or progression. Indeed, a number of MAPs have been described as oncogenes or tumor suppressors whose deregulation has a major impact on cancer aggressiveness and clinical outcome for the patients. In this review we will focus on microtubule-associated proteins with tumor suppressor effects (MATS) in breast cancer.

A family of microtubule-associated tumor suppressors (MATS) in breast cancer

A total of 10 MAPs with tumor suppressive functions have been identified in breast cancer. These (RASSF1A), and Von Hippel-Lindau (VHL) (Table 1).

BRCA1 is known as a master tumor suppressor in breast cancer. Interestingly, besides its key role in DNA repair, BRCA1 has also been shown to reduce microtubule dynamics [START_REF] Sung | BRCA1 regulates microtubule dynamics and taxane-induced apoptotic cell signaling[END_REF]. BRCA1 was found to localize on the mitotic spindle -tubulin, a centrosomal protein crucial for microtubule nucleation [START_REF] Hsu | BRCA1 is associated with the centrosome during mitosis[END_REF][START_REF] Lotti | Subcellular localization of the BRCA1 gene product in mitotic cells[END_REF]. APC, another master tumor suppressor, also localizes at the centrosome [START_REF] Louie | Adenomatous polyposis coli and EB1 localize in close proximity of the mother centriole and EB1 is a functional component of centrosomes[END_REF], a localization shared by other MATS such as ATIP3 [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], RASSF1A [START_REF] Liu | Control of microtubule stability by the RASSF1A tumor suppressor[END_REF][START_REF] Rong | Tumor suppressor RASSF1A is a microtubule-binding protein that stabilizes microtubules and induces G2/M arrest[END_REF] and VHL [START_REF] Thoma | VHL loss causes spindle misorientation and chromosome instability[END_REF]. Of note, APC was the first protein shown to interact with microtubule end-binding protein EB1 [START_REF] Morrison | The APC-EB1 interaction[END_REF] via a canonical SxIP motif [START_REF] Akhmanova | Control of microtubule organization and dynamics: two ends in the limelight[END_REF][START_REF] Honnappa | An EB1-binding motif acts as a microtubule tip localization signal[END_REF][START_REF] Slep | Structural determinants for EB1-mediated recruitment of APC and spectraplakins to the microtubule plus end[END_REF]. APC was thus identified as a plus-end tracking protein [START_REF] Slep | Structural determinants for EB1-mediated recruitment of APC and spectraplakins to the microtubule plus end[END_REF], like NAV3 [START_REF] Cohen-Dvashi | Navigator-3, a modulator of cell migration, may act as a suppressor of breast cancer progression[END_REF] and CYLD [START_REF] Gao | The tumor suppressor CYLD regulates microtubule dynamics and plays a role in cell migration[END_REF] that bind EB1 through SxIP and CAP-Gly motifs, respectively. In addition, most MATS were shown to decorate the microtubule lattice in interphase and/or the mitotic spindle during mitosis [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] Liu | Control of microtubule stability by the RASSF1A tumor suppressor[END_REF][START_REF] Rong | Tumor suppressor RASSF1A is a microtubule-binding protein that stabilizes microtubules and induces G2/M arrest[END_REF][START_REF] Thoma | VHL loss causes spindle misorientation and chromosome instability[END_REF][START_REF] Munemitsu | The APC gene product associates with microtubules in vivo and promotes their assembly in vitro[END_REF][START_REF] Stegmeier | The tumor suppressor CYLD regulates entry into mitosis[END_REF][START_REF] Ishii | FEZ1/LZTS1 gene at 8p22 suppresses cancer cell growth and regulates mitosis[END_REF][START_REF] Muranen | The tumor suppressor merlin interacts with microtubules and modulates Schwann cell microtubule cytoskeleton[END_REF] (Table 1).

The majority of MATS are microtubule stabilizers that regulate different parameters of microtubule dynamics. Indeed, ATIP3 [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], BRCA1 [START_REF] Sung | BRCA1 regulates microtubule dynamics and taxane-induced apoptotic cell signaling[END_REF], Merlin [START_REF] Smole | Tumor suppressor NF2/Merlin is a microtubule stabilizer[END_REF] and RASSF1A [START_REF] Liu | Control of microtubule stability by the RASSF1A tumor suppressor[END_REF][START_REF] Rong | Tumor suppressor RASSF1A is a microtubule-binding protein that stabilizes microtubules and induces G2/M arrest[END_REF] decrease microtubule growth rate whereas APC [START_REF] Slep | Structural determinants for EB1-mediated recruitment of APC and spectraplakins to the microtubule plus end[END_REF] and VHL [START_REF] Thoma | VHL loss causes spindle misorientation and chromosome instability[END_REF] protect MTs from depolymerization. FHIT [START_REF] Chaudhuri | The tumor suppressor protein Fhit. A novel interaction with tubulin[END_REF] and LZTS1 [START_REF] Ishii | FEZ1/LZTS1 gene at 8p22 suppresses cancer cell growth and regulates mitosis[END_REF] promote MT assembly. NAV3 [START_REF] Cohen-Dvashi | Navigator-3, a modulator of cell migration, may act as a suppressor of breast cancer progression[END_REF] and CYLD [START_REF] Gao | The tumor suppressor CYLD regulates microtubule dynamics and plays a role in cell migration[END_REF] both stabilize microtubules and promote their assembly (Table 1). Considering that these proteins are often co-localized on microtubules and that they all contribute to the regulation of microtubule organization and dynamics, it will be of interest to evaluate whether different MATS may have compensatory effects -in which the loss of one of these proteins could be balanced by another -or cooperative effects -in which expression of two or more proteins is necessary for microtubule regulation.

In breast cancer, MATS have been shown to be downregulated in 30 to 80% of tumor samples compared to normal adjacent tissues [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] Cohen-Dvashi | Navigator-3, a modulator of cell migration, may act as a suppressor of breast cancer progression[END_REF][START_REF] Ho | Reduced expression of APC and DCC gene protein in breast cancer[END_REF][START_REF] Rakha | Expression of BRCA1 protein in breast cancer and its prognostic significance[END_REF][START_REF] Hayashi | Clinical significance of CYLD downregulation in breast cancer[END_REF][START_REF] Campiglio | FHIT loss of function in human primary breast cancer correlates with advanced stage of the disease[END_REF][START_REF] Lovat | LZTS1 downregulation confers paclitaxel resistance and is associated with worse prognosis in breast cancer[END_REF][START_REF] Morrow | Loss of tumor suppressor Merlin in advanced breast cancer is due to post-translational regulation[END_REF][START_REF] Hagrass | Methylation status and protein expression of RASSF1A in breast cancer patients[END_REF][START_REF] Zia | The expression of the von Hippel-Lindau gene product and its impact on invasiveness of human breast cancer cells[END_REF][START_REF] Hu | Novel insight into KLF4 proteolytic regulation in estrogen receptor signaling and breast carcinogenesis[END_REF] (Table 2). Except for BRCA1 and APC, MATS genes are not heavily mutated in primary breast tumors. Their reduced expression level in breast cancer results from promoter hypermethylation for APC, FHIT, LZTS1 and RASSF1A genes [START_REF] Hagrass | Methylation status and protein expression of RASSF1A in breast cancer patients[END_REF][START_REF] Virmani | Aberrant methylation of the adenomatous polyposis coli (APC) gene promoter 1A in breast and lung carcinomas[END_REF][START_REF] Yang | Two-hit inactivation of FHIT by loss of heterozygosity and hypermethylation in breast cancer[END_REF][START_REF] Chen | Down-regulation of tumor suppressor gene FEZ1/LZTS1 in breast carcinoma involves promoter methylation and associates with metastasis[END_REF] and loss of heterozygosity for APC, BRCA1 and FHIT [START_REF] Virmani | Aberrant methylation of the adenomatous polyposis coli (APC) gene promoter 1A in breast and lung carcinomas[END_REF][START_REF] Yang | Two-hit inactivation of FHIT by loss of heterozygosity and hypermethylation in breast cancer[END_REF][START_REF] Rosen | BRCA1 gene in breast cancer[END_REF]. Other mechanisms involving microRNAmediated targeting of CYLD, LZTS1 and VHL genes in breast cancer have been more recently reported [START_REF] Kong | Upregulation of miRNA-155 promotes tumour angiogenesis by targeting VHL and is associated with poor prognosis and triple-negative breast cancer[END_REF][START_REF] Song | MicroRNA-301b promotes cell proliferation and apoptosis resistance in triple-negative breast cancer by targeting CYLD[END_REF][START_REF] Hou | miR-1207-5p regulates the sensitivity of triple-negative breast cancer cells to Taxol treatment via the suppression of LZTS1 expression[END_REF]. NF2 has been shown to be down-regulated at the post-translational level in advanced breast cancer by proteasome-dependent degradation [START_REF] Morrow | Loss of tumor suppressor Merlin in advanced breast cancer is due to post-translational regulation[END_REF]. The mechanisms responsible for down-regulation of MTUS1 and NAV3 genes in breast cancer still remain to be determined.

Interestingly, low levels of MTUS1/ATIP3 [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], CYLD [START_REF] Hayashi | Clinical significance of CYLD downregulation in breast cancer[END_REF], FHIT [START_REF] Yang | Two-hit inactivation of FHIT by loss of heterozygosity and hypermethylation in breast cancer[END_REF], NAV3 [START_REF] Cohen-Dvashi | Navigator-3, a modulator of cell migration, may act as a suppressor of breast cancer progression[END_REF] and RASSF1A [START_REF] Hagrass | Methylation status and protein expression of RASSF1A in breast cancer patients[END_REF] are significantly associated with ER-negative, highly proliferative and/or high-grade breast tumors, indicating that loss of MATS in breast cancer is associated with tumor aggressiveness. Furthermore, when considering MATS level and patient outcomes, studies have shown that for the majority of MATS, loss of expression is associated with reduced overall survival as well as reduced relapse free survival [START_REF] Cohen-Dvashi | Navigator-3, a modulator of cell migration, may act as a suppressor of breast cancer progression[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] Hayashi | Clinical significance of CYLD downregulation in breast cancer[END_REF][START_REF] Lovat | LZTS1 downregulation confers paclitaxel resistance and is associated with worse prognosis in breast cancer[END_REF][START_REF] Zia | The expression of the von Hippel-Lindau gene product and its impact on invasiveness of human breast cancer cells[END_REF][START_REF] Huszno | BRCA1 mutation in breast cancer patients: Analysis of prognostic factors and survival[END_REF][START_REF] Ginestier | Loss of FHIT protein expression is a marker of adverse evolution in good prognosis localized breast cancer[END_REF][START_REF] Martins | High RASSF1A promoter methylation levels are predictive of poor prognosis in fine-needle aspirate washings of breast cancer lesions[END_REF] (Table 2). Thus, MATS may represent useful prognostic biomarkers in breast cancer.

Prognostic value of ATIP3/EB1 combination : two biomarkers are better than one

The ATIP3 protein is an interesting example of a MATS with prognostic value in breast cancer. ATIP3

belongs to a family of proteins encoded by alternative splicing of the MTUS1 gene located at chromosome 8p22 [START_REF] Benedetto | Structural organization and expression of human MTUS1, a candidate 8p22 tumor suppressor gene encoding a family of angiotensin II AT2 receptor-interacting proteins[END_REF][START_REF] Rodrigues-Ferreira | An ATIPical family of angiotensin II AT2 receptorinteracting proteins[END_REF]. This protein is expressed in epithelial cells of the mammary gland in normal breast tissue and is markedly down-regulated both at the mRNA and protein level in half of invasive breast tumors [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]. Low levels of ATIP3 are associated with tumor aggressiveness, metastatic properties and reduced clinical outcome for breast cancer patients. Furthermore, re-expression of ATIP3 into breast cancer cell lines significantly reduces cell proliferation, polarity and migration, as well as tumor growth and metastasis in experimental mouse models [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], highlighting ATIP3 as a prognostic biomarker and a potent anti-cancer and anti-metastasis protein.

As mentioned above, ATIP3 localizes at the centrosome and along the microtubule lattice in interphase and decorates the mitotic spindle and spindle poles during mitosis [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]. Proximity mapping and functional analysis of the human centrosome-cilium interactome also revealed that ATIP3/MTUS1 belongs to a centrosomal protein network that regulates centrosomal functions [START_REF] Gupta | Dynamic protein interaction landscape of the human centrosome-cilium interface[END_REF]. Like most other MATS (Table 1), ATIP3 stabilizes microtubules. Its depletion significantly increases microtubule dynamics at the plus end by increasing microtubule growth and growth rate and reducing the time spent in pause [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]. At the molecular level, ATIP3 directly interacts with end-binding protein EB1 via a proline-rich and basic SxIP-like motif (RPLPLP) [START_REF] Velot | Negative regulation of EB1 turnover at microtubule plus ends by interaction with microtubule-associated protein ATIP3[END_REF]. ATIP3-EB1 interaction takes place in the cytosol, is independent of microtubules, and impairs EB1 binding and turnover at the plus ends [START_REF] Velot | Negative regulation of EB1 turnover at microtubule plus ends by interaction with microtubule-associated protein ATIP3[END_REF]. Strikingly, while almost all SxIP-containing EB1-binding proteins are +TIPs that localize at the plus end [START_REF] Akhmanova | Control of microtubule organization and dynamics: two ends in the limelight[END_REF][START_REF] Jiang | A Proteome-wide screen for mammalian SxIP motif-containing microtubule plus-end tracking proteins[END_REF][START_REF] Honnappa | An EB1-binding motif acts as a microtubule tip localization signal[END_REF], ATIP3 is a unique example of SxIP-containing protein that does not accumulate at the plus ends and rather acts as an endogenous antagonist of EB1 [START_REF] Nehlig | Regulation of end-binding protein EB1 in the control of microtubule dynamics[END_REF].

In breast cancer, EB1 levels were found elevated at the mRNA and protein levels compared to normal tissue [START_REF] Dong | Oncogenic function of microtubule endbinding protein 1 in breast cancer[END_REF][START_REF] Rodrigues-Ferreira | Combinatorial expression of microtubule-associated EB1 and ATIP3 biomarkers improves breast cancer prognosis[END_REF]. Furthermore, EB1 (but not EB2 or EB3) was identified as a prognostic biomarker whose high expression in breast tumors associates with tumor malignancy, high histological grade and reduced overall survival of the patients [START_REF] Rodrigues-Ferreira | Combinatorial expression of microtubule-associated EB1 and ATIP3 biomarkers improves breast cancer prognosis[END_REF]. The observation that ATIP3 negatively regulates EB1 functions launched further studies to investigate the clinical relevance of ATIP3-EB1 interaction for breast cancer patients. Results showed that in high-EB1 expressing breast tumors, ATIP3 deficiency results in increased tumor aggressiveness, with subsequent consequences on patient prognosis (Figure 1).

Furthermore, analyzing combinatorial expression of ATIP3 and EB1 in five independent cohorts of patients significantly improved breast cancer diagnosis and prognosis compared to each biomarker alone [START_REF] Rodrigues-Ferreira | Combinatorial expression of microtubule-associated EB1 and ATIP3 biomarkers improves breast cancer prognosis[END_REF]. Importantly, these studies allowed to identify a population of breast cancer patients of worse clinical outcome expressing high EB1 and low-ATIP3 levels. This population of patients, that represents 30% of breast cancer cases, may be eligible to molecular therapies aimed at restoring or mimicking ATIP3-EB1 interaction. Integrative approaches have recently been described for the discovery of small molecule modulators of EB1 binding to SxIP motifs [START_REF] Almeida | Targeting SxIP-EB1 interaction: an integrated approach to the discovery of small molecule modulators of dynamic binding sites[END_REF]. Such strategies may open interesting perspectives towards the development of personalized treatments to compensate for ATIP3 loss in a selected population of patients with high-EB1/low-ATIP3 expressing breast tumors.

Notably, other MATS such as APC, CYLD and NAV3 also interact with EB1 (Table 1). However, in contrast to ATIP3, these MATS are +TIPs that accumulate at the microtubule plus ends. Furthermore, APC and CYLD have been shown to cooperate with EB1 rather than antagonize its effects on cell migration [START_REF] Wen | EB1 and APC bind to mDia to stabilize microtubules downstream of Rho and promote cell migration[END_REF][START_REF] Green | APC and EB1 function together in mitosis to regulate spindle dynamics and chromosome alignment[END_REF][START_REF] Li | CYLD coordinates with EB1 to regulate microtubule dynamics and cell migration[END_REF]. These observations may suggest that loss of APC or CYLD in high-EB1 expressing breast tumors may impact tumor aggressiveness and clinical outcome for the patients. It will be of interest to investigate whether, similar to the ATIP3-EB1 couple, combinatorial expression of CYLD-EB1 and/or APC-EB1 may improve breast cancer prognosis.

Concluding remarks

In conclusion, among the large number of MAPs that control the microtubule network in interphase and mitosis, only few have been identified as bona fide microtubule-associated tumor suppressors (MATS) in breast cancer. Their deficiency in breast tumors is associated with poor prognosis of the patients, highlighting their potential value as biomarkers. Pioneer studies of ATIP3, a MATS that binds to EB1 and antagonizes its oncogenic effects in breast cancer, provide the first proof-of-concept that combinatorial expression of two biomarkers with opposite actions on microtubules improves breast cancer prognosis compared to each biomarker alone.

Several MATS do co-localize inside the cell, and some of them have been shown to act in concert. This raises the possibility that combinatorial expression of those microtubule-associated tumor suppressors may have improved value as biomarkers for the stratification of breast cancer patients in the context of personalized medicine. These data are particularly important considering increasing evidence that large numbers of MAPs are engaged into protein networks whose coordinated actions allow fine tuning of microtubule dynamics and assembly. In this line, the concomitant loss of BRCA1 and FHIT in sporadic breast cancer has been associated with reduced patients survival, suggesting that combination of these two MATS may be used as a marker to identify a subpopulation of breast tumor with poor prognosis [START_REF] Soares | Concomitant loss of heterozygosity at the BRCA1 and FHIT genes as a prognostic factor in sporadic breast cancer[END_REF]. VHL nc [START_REF] Zia | The expression of the von Hippel-Lindau gene product and its impact on invasiveness of human breast cancer cells[END_REF][START_REF] Hu | Novel insight into KLF4 proteolytic regulation in estrogen receptor signaling and breast carcinogenesis[END_REF] MicroRNA targeting [START_REF] Kong | Upregulation of miRNA-155 promotes tumour angiogenesis by targeting VHL and is associated with poor prognosis and triple-negative breast cancer[END_REF] [37]

  proteins include Adenomatous Polyposis Coli (APC), Microtubule-associated tumor suppressor 1 (MTUS1) protein ATIP3, Breast Cancer 1 (BRCA1), Cylindromatosis tumor suppressor (CYLD), Fragile Histidine Triad (FHIT), Leucine Zipper putative Tumor Suppressor 1 (LZTS1), Neurofibromatosis 2 (NF2) protein Merlin, Navigator-3 (NAV3), RAS Association domain Family 1A

Legend to Figure 1 .

 1 Further studies are warranted to evaluate the prognostic value of combined alterations of two or more MATS, together with other MAPs and their binding partners in breast cancer. The high complexity of MAPs networks suggests a novel paradigm for future discovery of a new generation of complex prognostic biomarkers in breast cancer.Two biomarkers are better than one. Consequences of combinatorial expression of EB1 and ATIP3on microtubule dynamics at the plus end (upper panel), breast cancer aggressiveness (lower panel) and overall survival of breast cancer patients (right panel). Tumors were classified according to combined EB1-ATIP3 expression as group 1 (EB1-low and ATIP3-low), group 2 (EB1-low and ATIP3-high), group 3 (EB1-high and ATIP3-low) and group 4 (EB1-high and ATIP3-high). Kaplan-Meier representation on the right indicates that breast tumors from group 3 are of worse prognosis compared to all other groups (p<0.05).

Table 1 . Microtubule-Associated Tumor Suppressors (MATS) properties related to microtubules.

 1 Protein localization, effect on microtubule assembly and stability, and ability to interact with EB1, are presented. nd : not determined

	MATS	Name	Localization	Effect on MT	EB1 interaction
	APC	Adenomatous Polyposis Coli	MT network, centrosome, MT plus ends [13, 23]	Stabilization [20]	SxIP [19, 20]
	ATIP3	Microtubule associated tumor suppressor MTUS1 /ATIP3	MT network, centrosome, mitotic spindle [14, 27]	Stabilization [27]	RPLP [52]
	BRCA1	Breast Cancer 1	Centrosome, mitotic spindle [11, 12]	Stabilization [10]	no
	CYLD	Cylindromatosis tumor suppressor	MT network, MT plus end, midbody [22, 24]	Stabilization and assembly [22]	CAP-Gly [59]
	FHIT	Fragile Histidine Triad	nd	Tubulin interaction, MT assembly [29]	nd
	LZTS1	Leucine Zipper putative Tumor Suppressor 1	MT network [25]	MT assembly [25]	nd
	Merlin/NF2	Neurofibromatosis 2 (NF2) protein Merlin	MT network, mitotic spindle [26]	Stabilization [28]	nd
	NAV3	Navigator-3	MT plus end [21]	Stabilization [21]	SxIP [8]
	RASSF1A	RAS Association domain Family 1A	MT network, centrosome, mitotic spindle [15, 16]	Stabilization [15, 16]	nd
	VHL	Von Hippel-Lindau	MT network, centrosome, mitotic spindle [17]	Stabilization [17]	nd

Table 2 . Microtubule-Associated Tumor Suppressors (MATS) in breast cancer.

 2 The percentage of primary breast tumors with reduced expression of MATS, mechanisms of gene inactivation, and prognostic value of MATS in breast cancer patients, are presented. LOH: Loss Of Heterozygosity; nc : not calculated; nd : not determined. A star indicates down-regulation at the posttranslational level.

Acknowledgements : This work has been funded by Gustave Roussy Cancer Center, the ANR grant MMO ANR-10-IBHU-0001, the Comité Ile-de-France of the Ligue Nationale contre le Cancer, the Ligue contre le Cancer 94/Val-de-Marne, the Entreprises contre le cancer (GEFLUC) Ile-de-France, the Fondation ARC pour la recherche contre le cancer, the CNRS, the INSERM, the Foundation Janssen Horizon, the Fonds de Dotation Agnès b., the association Odyssea and Prolific.

Conflict of interests :

Authors declare that they have no conflict of interests.


Lolita Portal

Didier Morin

Roberto Motterlini

Bijan Ghaleh

Sandrine Pons

The CO-releasing molecule CORM-3 protects adult cardiomyocytes against hypoxia-reoxygenation by modulating pH restoration

Keywords: hypoxia-reoxygenation, cardiomyocytes, carbon monoxide, pH hypoxia-reoxygenation, cardiomyocytes, carbon monoxide, pH

Several studies have reported that CORM-3, a water-soluble carbon monoxide releasing molecule, elicits cardioprotection against myocardial infarction but the mechanism remains to be investigated. Numerous reports indicate that inhibition of pH regulators, the Na + /H + exchanger (NHE) and Na + /HCO3 -symporter (NBC), protect cardiomyocytes from hypoxia/reoxygenation injury by delaying the intracellular pH (pHi) recovery at reperfusion. Our goal was to explore whether CORM-3-mediated cytoprotection involves the modulation of pH regulation. When added at reoxygenation, CORM-3 (50 µM) reduced the mortality of cardiomyocytes exposed to 3h of hypoxia and 2h of reoxygenation in HCO3 --buffered solution. This effect was lost when using inactive iCORM-3, which is depleted of CO and used as control, thus implicating CO as the mediator of this cardioprotection. Interestingly, the cardioprotective effect of CORM-3 was abolished by switching to a bicarbonate-free medium. This effect of CORM-3 was also inhibited by 5-hydroxydecanoate, a mitochondrial ATP-dependent K + (mKATP) channel inhibitor (500 µM) or PD098059, a MEK1/2 inhibitor (10 µM). In additional experiments and in the absence of hypoxia-reoxygenation, intracellular pH was monitored in cardiomyocytes exposed to cariporide to block NHE activity. CORM-3 inhibited alkalinisation and this effect was blocked by PD098059 and 5-HD. In conclusion, CORM-3 protects the cardiomyocyte against hypoxia-reoxygenation injury by inhibiting a bicarbonate transporter at reoxygenation, probably the Na + /HCO3 -symporter. This cardioprotective effect of CORM-3 requires the activation of mKATP channels and the activation of MEK1/2.

hypoxia-reoxygenation by modulating pH restoration. [START_REF] Yamamoto | Enhanced activity of ventricular Na+-HCO3-cotransport in pressure overload hypertrophy[END_REF].51 e-mail: bijan.ghaleh@inserm.fr

INTRODUCTION

Small amount of carbon monoxide (CO) gas is continuously produced in mammals via the endogenous degradation of heme by a family of constitutive (HO-2) and inductible (HO-1) heme oxygenase enzymes [START_REF] Coburn | Endogenous carbon monoxide production in man[END_REF]. Several studies demonstrated the anti-apoptotic and cardioprotective properties of the endogenous HO-1/CO system [START_REF] Brouard | Carbon monoxide generated by heme oxygenase 1 suppresses endothelial cell apoptosis[END_REF][START_REF] Wang | Cardioprotective and antiapoptotic effects of heme oxygenase-1 in the failing heart[END_REF]. In this context, Motterlini et al. [START_REF] Motterlini | Carbon monoxide-releasing molecules: characterization of biochemical and vascular activities[END_REF] have developed a group of compounds known as CO-releasing molecules (CO-RMs) that mimic the HO-1/CO pathway by liberating CO under appropriate conditions in biological systems. Among them, CORM-3, a water-soluble CO-releasing molecule, elicits cardioprotection through a mechanism involving mitochondrial ATPdependent potassium channels (mKATP) [START_REF] Clark | Cardioprotective actions by a water-soluble carbon monoxide-releasing molecule[END_REF]. This was confirmed in vivo as an intravenous infusion of CORM-3 significantly reduced infarct size in mice subjected to coronary artery occlusion [START_REF] Guo | Administration of a CO-releasing molecule at the time of reperfusion reduces infarct size in vivo[END_REF]. However, the mechanism of its beneficial action remains to be investigated.

It is well known that reperfusion injury is partly due to a rapid normalization of intracellular pH (pHi) during revascularisation [START_REF] Inserte | Effect of acidic reperfusion on prolongation of intracellular acidosis and myocardial salvage[END_REF]. This pHi recovery involves the activation of two major transporters, the Na + /H + exchanger (NHE) and the Na + /HCO3 -cotransporter (NBC) [START_REF] Vandenberg | Mechanisms of pHi recovery after global ischemia in the perfused heart[END_REF]. Inhibition of NHE by cariporide has been shown to protect cardiomyocytes by delaying pHi recovery at the time of reperfusion [START_REF] Xiao | Activity of the Na(+)/H(+) exchanger is critical to reperfusion damage and preconditioning in the isolated rat heart[END_REF]. Inhibition of NBC also affords cardioprotection [START_REF] Khandoudi | Inhibition of the cardiac electrogenic sodium bicarbonate cotransporter reduces ischemic injury[END_REF]. In this setting, previous studies demonstrated that prolonged acidosis at reperfusion is protective by inhibiting mitochondrial permeability transition pore (mPTP), reducing calcium overload and excessive contractile activation [START_REF] Orchard | Effects of changes of pH on the contractile function of cardiac muscle[END_REF][START_REF] Ruiz-Meana | Glycine protects cardiomyocytes against lethal reoxygenation injury by inhibiting mitochondrial permeability transition[END_REF][START_REF] Spitzer | Intracellular pH regulation in heart[END_REF]. Concomitantly, during reperfusion, survival signaling pathways may be triggered and among them, the activation of RISK (Reperfusion Injury Salvage Kinase) pathway including cascades of protein kinases (e.g., ERK1/2, p38 kinase, Akt) or mKATP channels are involved. Several studies suggested a link between regulation of pHi transporters, prolonged acidosis and activation of these survival kinase pathways [START_REF] Cohen | Acidosis, oxygen, and interference with mitochondrial permeability transition pore formation in the early minutes of reperfusion are critical to postconditioning's success[END_REF][START_REF] Fujita | Prolonged transient acidosis during early reperfusion contributes to the cardioprotective effects of postconditioning[END_REF], while other reports indicate that ERK1/2 is activated following a transient production of ROS and participates to the regulation of NBC [START_REF] Baetz | The ERK pathway regulates Na(+)-HCO(3)(-) cotransport activity in adult rat cardiomyocytes[END_REF][START_REF] Giusti | Role of reactive oxygen species (ROS) in angiotensin II-induced stimulation of the cardiac Na+/HCO3-cotransport[END_REF][START_REF] Giusti | Angiotensin II inhibits the electrogenic Na+/HCO3-cotransport of cat cardiac myocytes[END_REF]. Accordingly, the aim of this study was to explore whether the cytoprotective effect of CORM-3 involves modulation of pH at reoxygenation in a model of hypoxiareoxygenation and whether it requires MEK1/2 that is upstream of ERK1/2, p38 pathways and mKATP channels.

METHODS

Animals

Male 8 to 12 week-old wild-type C57BL/6J mice (n=37) (R. Janvier, Le Genest St Isle, France) were housed in an air-conditioned room with a 12 h light-dark cycle and received standard rodent chow and drinking water ad libitum. The authors have been granted a license from the institutional office "Préfecture du Val de Marne" (France) to conduct animal research (agreement A94-028-028). The investigations were performed in accordance with the European Directives (2010/63/EU).

Simulated ischemia-reoxygenation in adult mice cardiomyocytes

Adult mouse cardiomyocytes were isolated according to the technique previously described [START_REF] Portal | A model of hypoxia-reoxygenation on isolated adult cardiomyocytes: Charaterization, comparison with ischemia-reperfusion, and application to the cardioprotective effect of regular treadmill exercise[END_REF]. We obtained a cellular pellet which was washed 6 times with buffer containing 5% bovine calf serum and increasing concentrations of Ca 2+ . With this method, we routinely obtained a high percentage (80%) of rod-shaped myocytes with clear striations and without visible vesicles. Only one mouse (i.e., one individual isolation) was used for each set of experience. Cardiomyocytes obtained from different hearts were never pooled.

The freshly isolated cardiomyocytes were immediately re-suspended in serumfree/glucose-free medium, allowed to settle for 10 min and the supernatant was withdrawn and replaced with the serum-free/glucose-free medium bubbled with N2 (pH 7.4). Petri dishes (35 mm) were filled with 1 mL of this final cellular suspension and incubated for 3 hours in an O2/CO2 incubator containing a humidified atmosphere of 1% O2, 5% CO2 and 94% N2.

These conditions aimed at simulating ischemia [START_REF] Portal | A model of hypoxia-reoxygenation on isolated adult cardiomyocytes: Charaterization, comparison with ischemia-reperfusion, and application to the cardioprotective effect of regular treadmill exercise[END_REF]. This simulated ischemia will be referred as "hypoxia" throughout the text of this report. After hypoxia (3 h), cells were reoxygenated by adding 1 mL of a medium supplemented with bovine calf serum (10%) and glucose (11 mM) bubbled with O2 (i.e., final concentrations of 5% bovine calf serum and 5.5 mM glucose in 2 mL) and were placed under normoxia during 2 hours (CO2 incubator).

To provide control conditions, freshly isolated cardiomyocytes were washed twice with a medium supplemented with bovine calf serum (5%) and glucose (5.5 mM). Petri dishes were filled with 1 mL of this final cellular suspension and cells were placed under normoxia.

Reoxygenation was mimicked by adding 1 mL of the same medium. Experiments were performed at 37°C. Hypoxia-reoxygenation sequences were performed either in 1) HCO3 --containing medium (mM : NaCl 113, KCl 4.7, KH2PO4 0.6, Na2HPO4 0.6, MgSO4 1.2, NaHCO3 12, KHCO3 10, HEPES 10, taurine 30, CaCl2 1; 37°C); under these conditions, both pHi regulatory systems NHE and NBC are operative, or 2) in HCO3 --free medium (mM : NaCl 113, KCl 4.7, KH2PO4 0.6, Na2HPO4 0.6, MgSO4 1.2, HEPES 10, taurine 30, CaCl2 1 ; 37°C) which implies NBC inhibition.

CORM-3 or inactive CORM-3 (iCORM-3) (50 µM) was added at the time of reoxygenation. CORM-3 was synthetized as previously described [START_REF] Clark | Cardioprotective actions by a water-soluble carbon monoxide-releasing molecule[END_REF] and stock solutions (5 mM) were prepared freshly the day of the experiments by solubilizing the compound in distilled water. iCORM-3 was prepared by dissolving CORM-3 in phosphate buffered saline and allowing liberation of CO over 2 days at room temperature. Residual CO was eliminated by bubbling the previous solution with N2. The concentration of CORM-3 was defined as the concentration producing maximal protection during preliminary experiments investigating 25, 50 and 100 µM. This is was in agreement with a previous report showing cardioprotection with the concentration of 50 µM [START_REF] Clark | Cardioprotective actions by a water-soluble carbon monoxide-releasing molecule[END_REF].

To explore signalling pathways involved in cardioprotection of CORM-3, additional experiments were performed by exposing cells to reoxygenation in the presence of the mKATP channel inhibitor 5-HD (5-hydroxydecanoate, 500 µM), the MEK1/2 inhibitor PD098059 (10 µM) or the p38 kinase inhibitor SB202190 (10 µM).

Measurement of cell viability

At the end of the sequence of hypoxia-reoxygenation or normoxia, cardiomyocyte survival was measured using 0.4% trypan blue solution (Sigma, Saint Quentin Fallavier, France).

Cardiomyocytes were visualized under microscope. Ten fields were analyzed at ×20 magnification and at least 300 cells were counted in each dish. The number of viable (unstained) and non viable (blue stained) cells was recorded and the percentage of viability was defined as the ratio between number of unstained myocytes and total cells. Survival was expressed as the percentage of the normoxic control values which were set at 100%.

pHi measurements

Freshly isolated cardiomyocytes were plated in Petri dishes pre-coated with laminin (12.5 µg/mL) and containing 2 mL of HCO3 -medium supplemented with glucose and ITS (mg/mL: recombinant human insulin 10 -3 , human transferrin 0.55. 10 -3 , sodium selenite 0.5. 10 -6 ). Cells were allowed to attach for 2 h in humidified 5% CO2-95% air at 37°C and then washed once to remove unattached cells before being used. All experiments were carried out at 37°C. Adherent cardiomyocytes were incubated for 30 min with 5 µM BCECF-AM followed by washout. Dye-loaded cells were placed into a heated perfusion chamber (Warner Instruments Inc, Hamden, CT, USA) mounted on an Olympus IX-81 motorized fluorescent inverted microscope (Olympus, Rungis, France). BCECF fluorescence was detected using excitation wavelengths of 440 and 495 nm and a wavelength emission of 535 nm. Fluorescent images were acquired using ORCA-ER camera (Hamamatsu, Hamamatsu city, Japan). Fluorescence was integrated over a region of interest (≈ 200 µm 2 ) for each cardiomyocyte and the 495-to-440 nm fluorescence ratio was obtained using Cell-M software (Olympus, Rungis, France) [START_REF] Wu | DIDS-sensitive pHi regulation in single rat cardiac myocytes in nominally HCO3-free conditions[END_REF].

The fluorescence ratio was converted to pH by in situ calibration using the high K +nigericin method [START_REF] Wu | DIDS-sensitive pHi regulation in single rat cardiac myocytes in nominally HCO3-free conditions[END_REF]. The K + ionophore nigericin was used to collapse the H + gradient, so where R is the 495/440 nm fluorescence ratio; Rmax and Rmin are the maximum and minimum ratio values obtained at pH 9 and 4, respectively. The typical parameter values were 1.79 (Rmax), 0.37 (Rmin) and 7.26 (pKa).

that (K + i)/(K + o)= (H + i)/(H + o).

Ammonium pulse

As described above, the experiments were performed in HCO3 -buffered medium. Under this condition, both pHi regulatory systems NHE and NBC are operative. The activity of the two transporters was assessed by evaluating the pHi recovery from an ammonium prepulseinduced acute acid load [START_REF] Roos | Intracellular pH[END_REF]. Transient exposure (4 min) of cardiomyocytes to 30 mM NH4Cl followed by 20 min of washout with HCO3 -buffered medium (bubbled continuously with 5% CO2 and 95% O2, pH 7.4) was supplemented by several combinations of compounds.

Cariporide (30 µM) was added to block NHE activity. In these conditions, the effects of CORM-3 (50 µM) alone or combined with 5-HD (500 µM) and PD098059 (10 µM) were investigated. The intensity of pHi recovery was evaluated by a delta of pHi between the lower pHi value observed and the higher pHi value reached after the ammonium prepulse. Images were acquired every min during the 20 min of the pHi recovery period.

Statistics

All values are expressed as mean ± SEM. Comparisons were performed using a Kruskal-Wallis analysis followed by Mann-Whitney test. Non-parametric tests were chosen regarding the low number of samples. Bonferroni correction was applied to correct for multiple comparisons. Statistical significance was defined as a value of p<0.05.

RESULTS

Protective effects of CORM-3

The presence of CORM-3 reduced the mortality of cardiomyocytes exposed to 3h of hypoxia and 2h of reoxygenation in HCO3 --buffered solution (Figure 1A). The inactive iCORM-3 (50 µM), which is depleted of CO and used as negative control, did not protect cardiomyocytes against hypoxia-reoxygenation (HR) injury (absolute cell viability: 46±2% and 47±1% for HR and iCORM-3, respectively).

Bicarbonate-dependent cytoprotective effect of CORM-3

In this set of experiments, cardiomyocytes were incubated in HCO3 --free medium during the sequence of hypoxia-reoxygenation. Interestingly, the cardioprotective effect of CORM-3 was abolished by switching to the bicarbonate-free medium (Figure 1B).

The effect of CORM-3 on pHi recovery was investigated in cells superfused with HCO3 -buffered solution containing cariporide (Figure 2A). Following ammonium removal, while pHi recovered in the absence of cariporide, the delta of pHi recovery was significantly reduced by the addition of cariporide. When cells were exposed to cariporide plus CORM-3, this pHi recovery was almost abolished.

Signalling pathways involved in the protective effect of CORM-3

As shown in Figure 3A, the cardioprotective effect of CORM-3 was abolished by the presence of 5-HD. Similarly, the beneficial effect of CORM-3 on cardiomyocyte viability was significantly reduced by PD098059 (Figure 3B). We verified in additional experiments that 5-HD and PD098059 had no proper effects on cardiomyocyte submitted to HR (absolute cell viability: 49±4%, 51±1% vs 48±2% for PD098059 + HR and 5-HD + HR vs HR alone, respectively). Both HR+CORM-3-5HD and HR+CORM3-PD0908059 conditions were significantly different from corresponding HR+CORM3. In contrast, SB202190 did not modify cell viability (Figure 3C). SB202190 per se had no effect on cardiomyocyte viability (absolute cell viability vs HR: 49±2% vs 48±2%, respectively). We also verified that 5-HD, PH098059, SB202190 had no effect per se on cell viability during normoxia (absolute cell viability: 56±5%, 60±3%; 57±5%, 61±2 for normoxia per se, 5HD, SB202190 and PD098059, respectively Finally, cardiomyocytes were exposed to cariporide, CORM-3 plus 5-HD or PD098059

and pHi recovery at reoxygenation was evaluated (Figure 4). When cells were treated with 5-HD or PD098059 plus CORM-3 in the presence of cariporide, the change in pH was similar to control conditions, (absence of these inhibitors and the presence of cariporide).

DISCUSSION

Using an in vitro model of hypoxia-reoxygenation in adult mouse cardiomyocytes, we found that CORM-3 protected cells against reoxygenation damage at a concentration of 50 µM. The concentration of CORM-3 has been chosen based on a previous study which demonstrated the cytoprotection of CORM-3 against hypoxia-reoxygenation injury on rat H9C2 cardiac cells for this range of concentrations [START_REF] Clark | Cardioprotective actions by a water-soluble carbon monoxide-releasing molecule[END_REF]. The fact that the inactive compound (iCORM-3) did not show any protection against reoxygenation injury strongly emphasizes that CO liberated by CORM-3 is required to induce this cardioprotective effect.

In our experimental conditions, the cardioprotective effect of CORM-3 was abolished in the absence of bicarbonate in the extracellular medium indicating a bicarbonate-dependent protective mechanism, suggesting an effect on pH restoration at reoxygenation. As NHE usually operates in parallel with NBC, it was first necessary to inhibit the activity of NHE (i.e., a bicarbonate independent contributor) to better understand the effects of CORM-3 on pH restoration at reoxygenation. In these conditions, pHi recovery from an ammonium prepulse-induced acute acid load was almost abolished when cardiomyocytes were exposed to cariporide plus CORM-3. As cariporide inhibits NHE, it is reasonable to speculate that bicarbonate transporters as NBC are involved in the cardioprotection mediated by CORM-3.

Although not directly demonstrated, we believe that CORM-3 inhibited NBC, i.e., slowing the pHi recovery at reperfusion and thus protecting cardiomyocytes through inhibition of mPTP opening. Indeed, the regulation of pHi in cardiomyocytes depends on the activity of at least five transporters. NHE and NBC mediate acid-extrusion while Cl -/HCO3 -(CBE) and Cl - /OH -(CHE) exchanges mediate acid loading. A lactate/H + cotransport (monocarboxylate transporter, MCT) contributes to lactate washout at reperfusion [START_REF] Spitzer | Intracellular pH regulation in heart[END_REF]. The role of NHE in ischemia-reperfusion injury has been widely investigated. The inhibition of this exchanger has been shown to protect from contractile dysfunction in correlation with reduced cellular Na + and Ca 2+ levels [START_REF] Meng | Influence of perfusate pH on the postischemic recovery of cardiac contractile function: involvement of sodium-hydrogen exchange[END_REF][START_REF] Meng | Effect of amiloride and selected analogues on postischemic recovery of cardiac contractile function[END_REF][START_REF] Meng | Protective effects of 5-(N,N-dimethyl)amiloride on ischemia-reperfusion injury in hearts[END_REF].

The contribution of NBC to pHi recovery at reperfusion has been probably underestimated in several studies due to the use of HCO3 --free medium. Indeed, investigations performed in isolated hearts and cardiomyocytes demonstrated an important contribution of NBC to proton efflux at reperfusion [START_REF] Vandenberg | Mechanisms of pHi recovery after global ischemia in the perfused heart[END_REF]. Vandenberg et al. [START_REF] Vandenberg | Mechanisms of pHi recovery after global ischemia in the perfused heart[END_REF] reported pharmacological evidence suggesting that NBC contributed even more than NHE to the pHi recovery of reperfusion in ferret hearts. The coupling of NBC to Na + -coupled acid extrusion is of interest. Schafer et al.

[28] demonstrated in adult rat cardiomyocytes that simultaneous inhibition of NBC and NHE during reoxygenation prevented pHi recovery, attenuated Ca 2+ oscillations and abolished hypercontracture. Thus, NHE but also NBC contribute to Na + and Ca 2+ overload at reperfusion.

Ten Hove et al. [START_REF] Hove | Relative contributions of Na+/H+ exchange and Na+/HCO3-cotransport to ischemic Nai+ overload in isolated rat hearts[END_REF] showed that inhibition of one of these transporters did not increase the activity of the other one. Therefore, inhibition of only one transporter should be sufficient to slow the recovery of pHi at reperfusion and to induce cardioprotection. One study showed that mRNA levels of the electrogenic NBC form was increased by aortic constriction and that acid extrusion through NBC was increased in hypertrophied myocytes [START_REF] Yamamoto | Enhanced activity of ventricular Na+-HCO3-cotransport in pressure overload hypertrophy[END_REF]. Furthermore, overexpression of NBC (mRNA and proteins) has been demonstrated in human cardiomyopathy [START_REF] Khandoudi | Inhibition of the cardiac electrogenic sodium bicarbonate cotransporter reduces ischemic injury[END_REF]. This highlights NBC as an interesting target during ischemic disease.

Indeed, a previous study suggested that selective inhibition of NBC induces cardioprotection as Kandoudhi et al. [START_REF] Khandoudi | Inhibition of the cardiac electrogenic sodium bicarbonate cotransporter reduces ischemic injury[END_REF] demonstrated that administration of anti-NBC antibody to ischemicreperfused rat hearts markedly protects systolic and diastolic functions during reperfusion. It should be stressed that our study did not determined the exact nature of the transporter targeted by CORM-3. It should be noted that another HCO3 -dependent acid extruder, the Cl - /HCO3 -/Na + dependent cotransport (NDCBE or NBCn2) has been discovered in neonatal mouse heart and might participate to H + equivalent efflux [START_REF] Parker | The divergence, actions, roles, and relatives of sodiumcoupled bicarbonate transporters[END_REF].

This study also demonstrates that the effects of CORM-3 on bicarbonate-mediated pH recovery and hypoxia-reoxygenation viability involve mitochondrial KATP channels opening MEK1/2 and as a result probably ERK1/2 activity. Indeed, CORM-3 significantly reduces the variation of pH observed under cariporide (and vs control conditions), this means that 5-HD and PD098059 abolished the effects of CORM-3 on pH recovery. Indeed, we can speculate that CORM-3 triggered the opening of mKATP channels, which led to transitory mitochondrial ROS release in cytosol. This acted as the signalling molecules that induced the activation of MEK1/2 and subsequent inhibition of NBC. It should be acknowkedged that we did not use a selective inhibitor of mitochondrial KATP but the involvement of the KATP channels in the cardioprotective effect of CORM-3 was already demonstrated by Clark et al. [START_REF] Clark | Cardioprotective actions by a water-soluble carbon monoxide-releasing molecule[END_REF].

Furthermore, it has been demonstrated that CO activates KATP channels and that heme binding to a subunit of this protein is required for the CO-dependent increase in channel activity [START_REF] Khandoudi | Inhibition of the cardiac electrogenic sodium bicarbonate cotransporter reduces ischemic injury[END_REF].

Opening of mKATP channels is known to induce transient production of ROS by mitochondria [START_REF] Costa | The direct physiological effects of mitoK(ATP) opening on heart mitochondria[END_REF][START_REF] Hausenloy | Survival kinases in ischemic preconditioning and postconditioning[END_REF][START_REF] Pain | Opening of mitochondrial K(ATP) channels triggers the preconditioned state by generating free radicals[END_REF]. Furthermore, it has been demonstrated that CO-RMs and CO induce a transient and moderate production of mitochondrial ROS and particularly in cardiomyocytes [START_REF] Kondo-Nakamura | Brief exposure to carbon monoxide preconditions cardiomyogenic cells against apoptosis in ischemiareperfusion[END_REF][START_REF] Lancel | Carbon monoxide rescues mice from lethal sepsis by supporting mitochondrial energetic metabolism and activating mitochondrial biogenesis[END_REF][START_REF] Scragg | Carbon monoxide inhibits L-type Ca2+ channels via redox modulation of key cysteine residues by mitochondrial reactive oxygen species[END_REF][START_REF] Zuckerbraun | Carbon monoxide signals via inhibition of cytochrome c oxidase and generation of mitochondrial reactive oxygen species[END_REF]. In line with our proposed mechanism, several studies showed that ERK 1/2 is activated following a transient production of ROS and participates in the regulation of NBC [START_REF] Baetz | The ERK pathway regulates Na(+)-HCO(3)(-) cotransport activity in adult rat cardiomyocytes[END_REF][START_REF] Giusti | Role of reactive oxygen species (ROS) in angiotensin II-induced stimulation of the cardiac Na+/HCO3-cotransport[END_REF][START_REF] Giusti | Angiotensin II inhibits the electrogenic Na+/HCO3-cotransport of cat cardiac myocytes[END_REF]. Finally, p38 MAPK was not involved in the cytoprotective effects of CORM-3 as SB202190 did not modify cell viability. This kinase is known to regulate NBC and to be involved in the cytoprotective effect of CO but it was in a context of hepatic and pulmonary ischemia-reperfusion injury and not cardiac injury [START_REF] Amersi | Ex vivo exposure to carbon monoxide prevents hepatic ischemia/reperfusion injury through p38 MAP kinase pathway[END_REF][START_REF] Zhang | Carbon monoxide inhibition of apoptosis during ischemia-reperfusion lung injury is dependent on the p38 mitogen-activated protein kinase pathway and involves caspase 3[END_REF]. Finally, some limitations should be also acknowledged. The proper effects of CORM3, iCORM, 5HD and PD098059 on pHi were not investigated as our objective was to isolate the NBC activity independently from NHE. Some of our conclusions are also based only on the use of inhibitors and western blot experiments (e.g., MEK1/2 phosphorylation) would have strengthen this study.

In conclusion, CORM-3 protects cardiomyocytes against hypoxia-reoxygenation injury by inhibiting a bicarbonate transporter at reoxygenation, most likely the Na + /HCO3 - symporter. This cardioprotective effect of CORM-3 requires the activation of mitochondrial KATP channels and the activation of MEK1/2 pathway.

FIGURES LEGENDS

Min pHi

Min pHi

  Fluorescence ratio readings were taken for the cells exposed to high-K + medium (mM: KCl 140, MgCl2 1, 2,3-butanedione monoxime 10, nigericin 0.01) over a range of pH values obtained with one of the following organic buffers (mM): MES 20 (pH 4 to 6.5), HEPES 20 (pH 7 to 8.5) and CAPSO 20 (pH 9). A calibration curve was established by measuring the fluorescence ratio values between pHi 4 and 9. The response was linear for pHi 6.5 to 8 and followed the equation pHi=pKa-log((Rmax-R)/(R-Rmin))

Figure 1 :

 1 Figure 1: Effect of CORM-3 (50 µM) on cardiomyocyte survival in presence (panel A, n = 8)or absence (panel B, n= 6) of HCO3 -. Cardiomyocytes were exposed to 3 h hypoxia followed

Figure 2 :

 2 Figure 2: Effect of CORM-3 (50 µM) on cardiomyocyte intracellular pH (pHi) recovery from

Figure 3 :

 3 Figure 3: Effects of 5-hydroxydecanoate (5HD, panel A), PD098059 (panel B) and

Figure 4 :

 4 Figure 4: Effects of 5-hydroxydecanoate 5HD (panel A) and PD098059 (panel B) on the

Figure 4

 4 Figure 3

  Lolita Portal1 , Didier Morin 1 , Roberto Motterlini 2 , Bijan Ghaleh 1 , Sandrine Pons 1 .

	3-	U955-IMRB, Equipe 03, Inserm, UPEC, Ecole Nationale Vétérinaire d'Alfort, Créteil,
	France.
	4-	U955-IMRB, Equipe 12, Inserm, UPEC, Créteil, France
		Corresponding author:
		Pr Bijan Ghaleh
		INSERM U 955 Equipe 03
		Faculté de Médecine
		8 rue du Général Sarrail
		94000 Créteil
		Tel : 33-1-49.81.49.31
		Fax : 33-1-49.81.

ACKNOWLEDGEMENTS

This study was supported by a grant from the Fondation de France (2008-002688). Lolita Portal was a recipient of a grant from the Region Ile de France (CODDIM, 2010).

CONFLICT OF INTEREST

None.


J L Bouly

J C Curdy

R Geller

S V Golubev

A Lacoste

T Lamy

P Sole

P Sortais

S V Razin

J L Vieux-Rochaz

T Thuillier

A V Vodopyanov

V G Zorin

High current density production of multicharged ions with ECR plasma heated by gyrotron transmitter

In order to study the possibilities to produce high currents of pulsed heavy ion beams dedicated to synchrotron injection, two new approaches of ECR devices are now underway. The basic principle consists in maintaining a functioning point of the source with the highest density as possible and a minimum confinement time for the production of a given charge state. It means that for a constant n e i product we try to maximize n e and minimize i . For this purpose two experiments are in progress at ISN/Grenoble a and IAP/Nizhny Novgorod. b The first one consists of using a minimum ͉B͉ magnetic structure with a 1.8 mirror ratio characteristic value with a 28 GHz frequency injection. In this case we explore different functioning points up to 10 kW of UHF power. The second one consists of a simple mirror magnetic system ͑simple mirror ion source, SMIS͒ working at 37.5 GHz with a mirror ratio up to 3 ͑2.5 T͒ where we study discharges with a peak power up to 100 kW. We will show that, in spite of a very short rising time of the current, we can maintain the production of multicharged ions and that we can observe very high current densities. In the future, the challenge will be the design of an extraction geometry matching theses current levels.

I. INTRODUCTION

The two main parameters that measure the capability of a plasma to produce multicharged ion are the electronic density n e and the confinement time of the ions i especially if we assume that the suitable electronic energy can be achieved in any case due to the efficiency of the ECR heating.

The product n e i is a rough measurement of the collision rate so it is roughly proportional to the ͗Z͘ of the ion in the plasma. The ratio n e / i is roughly proportional to the flux of particles arriving to the wall so the current density of a given charge state.

The aim of this project consists in multiplying the density by a factor of 3, that seems achievable with a 28 GHz discharge and optimizing the confinement time with a divi-sion by a factor of 3. In this case the n e i product remains constant and only the ratio n e / i is multiplied by a factor of 10. The current is multiplied by a factor of 10 and the ͗Z͘ remains constant so we look for a modification of the charge state distribution as illustrated by Fig. 1. We think that this functioning point could be achieved in a quite simple 28 GHz classical source working with a mirror ratio around 1.5. Finally, if we plan to work with high density and low confinement it means that the loss rate of the particles will be very high so that will need of a strong UHF power density in order to maintain the electronic density.

II. PHOENIX 28 GHz

A. General layout

The purpose of this project consists of using a very high density plasma in a quite medium confinement device in order to generate very high current density beams for the generation of high currents. It is the reason why PHOENIX is a compact machine matched to a 10 kW, 28 GHz gyrotron. The plasma chamber is a 74 mm in diameter and 300 mm in length tube with a direct and axial injection of the wave. The waveguide is a TE01 oversized for the 28 GHz frequency and it has a diameter of 32 mm ͑Fig. 2͒.

An important point is the introduction of a thick insulator ͑3 mm͒ between the coils and the inner part of the source in order to achieve an insulation up to 60 kV for the extraction of high current density. The axial magnetic profile is just matched to a 28 GHz ECR frequency for a mirror ratio of roughly 1.5. In this case the maximum axial field is 1.6 T at the injection of the wave and 1.4 T at the extraction for a 1300 A current inside the coils. The electrical consumption is roughly 200 kVA in this case. The radial field is done with a classical 80 mm in diameter FeNdB hexapole delivering 1.5 T on the pole.

B. High current density measurements

In a first step we only try to identify the possibility of production of very high current densities ͑above 20 mAe/cm 2 ͒. We have started the source at the present maximum extraction voltage of 48 kV and with a 2 mm extraction. We have easily extracted 4 mAe of total current with nitrogen corresponding to 3 mAe of total current arriving on the Faraday cup ͑75% of transmission and 0.9 mAe of N 3ϩ ; Fig. 3͒. In this case it means that we can reach a density close to 100 mAe/cm 2 at least in a 2 mm hole.

In this case we have switched the extraction hole to 6 mm ͑gap 35 mm͒ and study the production and transmission at 45 kV ͑Fig. 4͒. We can see the currents delivered by the high voltage power supply, measured in a Faraday cup placed just at the exit of the puller, the sum of all the ionic currents arriving in the Faraday cup placed after the bending magnet and the current of 0 4ϩ as a function of the UHF power.

We can see that at a low extracted current of 3.5 mAe after the puller ͑12 mAe/cm 2 ͒ we have a very good transmission. All the drain current arrive at the exit of the puller and are transferred through the bending magnet. But around 6 mAe after the puller ͑21 mAe/cm 2 ͒ we observe a saturation of the current after the bending magnet ͑4.4 mAe͒ indicating a clear space charge limitation through the beam line. Now around 7 mAe after the puller we began to see a major difference between the drain current and the Faraday cup current indicating the beginning of current losses and generation of the secondary electrons directly on the puller. Now it is the extraction gap that is space charge limited.

The conclusion of the present experiments are that we can easily produce high current densities of multicharged ion but we have to match the extraction voltage to each value in order to get a good transmission. In a near future an upgrade of the voltage and the acceptance of the beam line will be done.

III. SMIS 37 GHz

A. General layout

In parallel with the PHOENIX experiment a second device developed at the IAP of Nizhny Novgorod ͑Russia͒ and called simple mirror ion source 37.5 GHz is used to study the production of very high current density of multicharged ions.

Here a new step in the reduction of the confinement is done. The source is reduced to a simple axial magnetic mirror placed around a simple stainless steel plasma chamber 1 ͑Fig. 5͒. There is no hexapole and the pulsed UHF power is injected on the axis of the source through a glass window. In this case the extraction hole is reduced to 1 mm only. Due to technological limitations the magnetic field is pulsed and has a maximum value of 2.5 T during roughly 10 ms. The pulsed duration of the UHF power is limited by the high voltage power supply and has a maximum duration of 1 ms with a peak power of 130 kW. Another specificity of this device is the use of a quasi-optical coupling for the UHF injection. The power extracted from the gyrotron is a Gaussian beam that is refocused by a dielectric lens before the injection under vacuum through the glass window of the source. In this case there is no physical connection between the transmitter and the source, so there is no problem of insulation and power density transmission.

The source can presently be biased only up to 15 kV and the analyze of the beam is done with the former charge breeding beam line of ISN/Grenoble that has been transferred to Nizhny Novgorod.

B. Preliminary result

We can see in Fig. 6 a first result obtained for nitrogen ͑with C from outgassing͒. Here we show the spectrum during the 1 ms pulse of the UHF power and at five steps of 200 s. We see that a full spectrum of multicharged ions of nitrogen can be produced in an opened trap. Here we have a clear illustration of the compensation of the reduction of the confinement time by a very strong power density used to maintain the electronic density. The peak power is 60 kW for a 1 l plasma chamber and the rising time of the current of N 4ϩ or N 5ϩ is only some hundreds of s. In this case the total current extract from the source is around 5 mAe with roughly 3 mAe measured at the level of the extraction Faraday cup. The current density reaches more than 400 mAe/cm 2 that is roughly 10 times more than with PHOENIX and 100 times than a classical source. Of course, the transmission is very poor because of the low extraction voltage and the poor acceptance of the beam line. This aspect will be upgraded in a near future but the purpose of this first experiment consists in showing the possibility to produce multicharged ions inside a ion source without radial magnetic field.

IV. CONCLUSION

We start new compact devices working with high frequency and high power density and we clearly observe a drastic increase of the current density of multicharged ions. These currents densities can be produced in a minimum ͉B͉ structure with a power density in the range of 5 kW per liter or in an open magnetic trap with a power density in the range of 50 kW per liter. The next step is now the control of the beam formation in the characteristic environment of an ECR ion source for the production of multi-milliamperes multicharged ion beams. 1 V. G. Zorin, S. V. Golubev, and S. V. Razin, Rev. Sci. Instrum. 69, 634 ͑1998͒.

a

  Under grant CERN/GSI/CEA/IN2P3 K598/PS and EU Contract No. HPRI-1999-50019. b Under Grant IAP/IN2P3-2/2001.

FIG. 1 .

 1 FIG. 1. Low flux and high flux charge state distribution with constant ͗Z͘.

FIG. 5 .

 5 FIG. 5. SMIS 37.5 GHz experimental setup.