domain: chim sdv

text:
Sarah Spreckelmeyer

Margot Van Der Zee

Benoît Bertrand

Ewen Bodio

Stefan Stürup

Angela Casini
email: casinia@cardiff.ac.uk

Benoîtbertrand

Prof Angela Casini

Relevance of Copper and Organic Cation Transporters in the Activity and Transport Mechanisms of an Anticancer Cyclometallated Gold(III) Compound in Comparison to Cisplatin

Keywords: organometallicgold compounds, cisplatin, cancer, drug transporters, organic cation transporters, copper transporters, ICP-MS

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Introduction

In the field of anticancer metallodrugs, platinum(II) compounds (cisplatin(Figure 1), carboplatin, oxaliplatin and nedaplatin) are still the gold-standard treatment for numerous types of cancers, 1 mostly applied in combination with other chemotherapeutics. However, their use is limited due to intrinsic or acquired resistance and severe side-effects, such as nephrotoxicity in the case of cisplatin. Consequently, a panel ofnon-platinum metallodrugs was designed as anticancer agents to achieve improved therapeutics. Many of them are in preclinical studies, but only a fewreached clinical trials and are still far from being marketed. [2][3][4] In general, the differential activity/toxicity profiles of anticancer drugs in different organs, such as the nephrotoxicity of cisplatin, may be related to different drug accumulation in the cells of these tissues. It is worth mentioning that higher drug accumulation in a particular cell type might be caused by either higher uptake or lower efflux 5,[START_REF] Russel | In Enzyme-and Transporter-Based Drug-Drug Interactions: Progress and Future Challenges[END_REF] , which emphasizes the need for mechanistic information on the drug transport mechanisms involved and possible interactions with membrane transporters. However, even for the well-established anticancer platinum drugs, knowledge about their mechanism of accumulation in cancer cells, as well as in healthy tissues, is incomplete and often contradictory. Specifically, the hOCT2 (human organic cation transporter 2) and hCTR1 (human copper transporter 1) have been postulated to be involved in the uptake of cisplatin, whereas the multidrug and toxic extrusion protein (MATE) and ATP7A/B seem to be involved in the drug's efflux, as recently reviewed by Spreckelmeyer et al. [START_REF] Spreckelmeyer | [END_REF] and others. 8,9 It should be noted that while hCTR1 is responsible for the cellular uptake of Cu + ions, the ATP7A and ATP7B are pumps known to be involved in the efflux of copper ions. MATEs are part of the organic cation homeostasis, belonging to the SLC47 family. In fact, MATEs act as H + /organic cation antiporters, transporting protons from the extracellular side to the cytoplasm in concomitance with organic cations export to the lumen of the proximal tubule. Several studies have confirmed cisplatin transport by MATEs. 10 Recently, Natile, Arnesano and co-workers investigated the binding of platinum(II) complexes to the Met-rich domain of hCTR1 by several methods including mass spectrometry and NMR spectroscopy. 11,12 According to the obtained results, cisplatin reacts with the copper binding domain forming adducts where all the original ligands of Pt(II) are substituted by the S-donor groups of Met side-chains. According to these observations, cisplatin would be actually sequestered by hCTR1 and not transported intracellularly. Furthermore, regulation of the expression of these membrane transporters upon metallodrug administration can also occur and its effects on cisplatin treatment should be considered. 13 Recently, Howell and coworkers reported on the use of the CRISPR-Cas9 technique to individually knock out the human copper transporters CTR1 and CTR2 and the copper chaperones ATOX1 and CCS in human cancer cells. 14 The obtained results showed that loss of such transporters does not produce a change in cisplatin sensitivity.

Among new generation metal-based complexes, gold(I) and gold(III) compounds raised interest in the last years showing promising anticancer properties in vitro, which prompted the investigation of their molecular mechanisms of biological action. 15,16 As a representative example, the gold(I) compound auranofin ([Au(I)(2,3,4,6-tetra-O-acetyl-1-(thio-S)--D-glucopyranosato)(triethylphosphine)]) is a potent cytotoxic agent in vitro and is currently in clinical trials. 17 Among the various families of gold compounds tested for their anticancer effects in the last decade, a variety of organometallic gold(I/III)appeared to be particularly promising. 18,19 Indeed, they feature potent anticancer activity in vitro, andare endowed with improved stability in physiological environment with respect to classical coordination complexes.In this context, our group has designed new gold(III) cyclometallated compounds featuring both bidentate C^N-and tridentate C^N^N-, C^N^C-, and N^C^N-donor ligands, with either five-or six-membered C,N rings. 15,[20][21][22][23][24][25] For example, a series of C^N gold(III) complexes of general formula[Au(py b -H)X2] (py b -H = C^N cyclometallated 2-benzylpyridine)has been developed by us showing interesting antiproliferative effects in a panel of human cancer cells in vitro. 26 While investigation of the possible pharmacological targets for this family of organometallic gold(III) complexes has led to the identification of a number of cancer-related proteins, including the selenoenzyme thioredoxin reductase, 27 and zinc finger proteins involved in DNA repair mechanisms, 28 among others, very scarce information is available concerning the drug transport mechanisms in cancer cells.As far as we are aware, only one in vitro study has been published evaluating the role of hOCTs, hCTR1 and of endocytotic processes in the uptake of a cytotoxic gold(I) NHC (N-heterocyclic carbene) complexin cancer cells. 29 The results suggest that cellular uptake for all tested gold complexes occurs mainly via the OCT transporters, but in one case also via hCTR1. In addition, some of the compounds were also internalized via the Na + /K + -dependent endocytosis. 29 In fact, while in general endocytosis is the vesicle-mediated process used by all cells to internalize extracellular molecules, some endocytotic processes depend on a sodium gradient and can be slowed down by adding ouabaine, an inhibitor of the Na + /K + pump.

More recently,we reported on the toxic effects and accumulation mechanisms of another C^N gold(III) complex -[Au(py b -H)(PTA)Cl] (PTA = 1,3,5-triazaphosphaadamantane)(Figure 1) -in healthy rat kidneys ex vivo, using the Precision Cut Tissue Slices (PCTS) method. 30 Interestingly, the obtained results seem to exclude OCTs-related uptake mechanisms as well as MATE efflux pathways for this gold(III) compound in kidneys. Here, we report on the synthesis and characterization of a novel cyclometallated C^N gold(III) compound (Figure 2, 1) [Au(py b -H)(PPh2Ar)Cl]PF6 (PPh2Ar = 3-[4-(diphenylphosphino)phenyl]-7methoxy-2H-chromen-2-one] featuring a coumarin ligand endowed with "smart"fluorescence properties for imaging in cells by fluorescence microscopy. [31][32][33] The smart character of this probe involves a dramatic drop of the fluorescence when the metal is released, allowing a reliable tracking of the complex: if strong fluorescence is observed it means that the "P-Au" bond is intact. The evaluation of the antiproliferative effects of the Au(III) complex compared to cisplatin was performed using a panel of human cancer cell lines as well as non-cancerous cells in vitro. Moreover, the involvement of different transporters (CTR1, OCT2, MATE, ATP7A/B) in the antiproliferative activity of the gold compound was studied via transporter inhibition experiments in comparison to cisplatin in two human ovarian cancer cell lines, one sensitive (A2780) and one resistant to cisplatin (A2780cisR). Furthermore, the accumulation and uptake mechanisms of the compounds werestudied in the same cells, evaluating the cellular metal content by Inductively Coupled Plasma Mass Spectrometry (ICP-MS).Preliminary fluorescence microscopy data allowed investigation of the subcellular localization of complex 1 in cancer cells.

Material and Methods

General Remarks

All reactions were carried out under an atmosphere of purified argon using Schlenk techniques. Solvents were dried and distilled under argon before use.Cisplatin was purchased at Sigma, while the precursor [Au(py b -H)Cl2] and the coumarin-phosphinehave been synthesized according to literature procedure. 34,33 The identity and purity (≥ 95%) of the gold complexes under investigation were unambiguously established using high-resolution mass spectrometry and NMR. All other reagents were commercially available and used as received. All the analyses were performed at the "Plateforme d'Analyses Chimiques et de Synthèse Moléculaire de l'Université de Bourgogne". Exact masses of the synthesized complexes were obtained on a Thermo LTQ Orbitrap XL. 1 H-(300.13, 500.13 or 600.23 MHz), 13 C-(125.77 or 150.90 MHz) and 31 P-(121.49, 202.45 or 242.94 MHz) NMR spectra were recorded on Bruker 300 Avance III, 500 Avance III or 600 Avance II spectrometers. Chemical shifts are quoted in ppm (δ)relative to TMS ( 1 H and 13 C) using the residual protonated solvent ( 1 H) or the deuterated solvent ( 13 C) as internal standards. 85% H3PO4 ( 31 P) was used as an external standard. Infrared spectra were recorded on a Bruker Vector 22 FT-IR spectrophotometer (Golden Gate ATR) and far infrared spectra were recorded on a Bruker Vertex 70v FT-IR spectrophotometer (Diamant ATR).

Synthesis of [Au(py b -H)(PPh2Ar)Cl].PF6 (1)

A round-bottom flask was charged with the precursor [Au(py b -H)Cl2] (50 mg, 0.115 mmol, 1eq.), KPF6 (106 mg, 0.573 mmol, 5 eq.) and 3-[4-(diphenylphosphino)phenyl]-7-methoxy-2Hchromen-2-one (PPh2Ar) (50 mg, 0.115 mmol, 1 eq.) in suspension into 5 mL of distilled acetone under argon atmosphere. Thegold complex precursor was solubilized after few minutes. The reaction was maintained at room temperature for 1.5 h; afterward, 10 mL of dichloromethane were added, and the yellow solution was filtrated through Celite® and concentrated under vacuum. The pure product was obtained after precipitation from a dichloromethane/pentane mixture as a yellow powder (91 mg, 0.092 mmol, 80 % yield). 1 H NMR (Acetone-d6, 500.13 MHz, 298 K): 3.97 (s, 3 H, OCH3), 4.49 (d, 1 H, 2 JH-H = 15.6 Hz, CH2-PyrBz), 5.05 (d, 1 H, 2 JH-H = 15.6 Hz, CH2-PyrBz), 6.52 (dt, 1 H, 3 JH-H = 8.5 Hz, 4 JH-H = 1.5 Hz, H 5' ), 6.88 (dd, 1 H, 3 JH-H = 7.5 Hz, 4 JH-H = 3.0 Hz, H 6' ), 6.98 (d, 1 H, 4 JH-H = 2.5 Hz, H D ), 7.00 (dd, 3 JH-H = 8.5 Hz, 4 JH-H = 2.5 Hz, H C ), 7.03 (dt, 3 JH-H = 8.5 Hz, 4 JH-H = 0.5 Hz, H 4' ), 7.33 (dd, 1 H, 3 JH-H = 8.5 Hz, 4 JH-H = 1.5 Hz, H 3' ), 7.60-7.66 (m, 4 H, Hortho-Ph), 7.70 (d, 3 JH-H = 8.5 Hz, H B ), 7.73-7.79 (m, 2 H, Hortho-pC6H4), 7.81 (t, 3 JH-H = 8.5 Hz, H 5 ), 7.89-8.01 (m, 8 H, Hmeta/para-Ph + Hmeta-pC6H4), 8.06 (d, 1 H, 3 JH-H = 8.5 Hz, H 3 ), 8.27 (s, 1 H, H A ), 8.31 (dt, 1 H, 3 JH-H = 8.5 Hz, 4 JH-H = 1.5 Hz, H 4 ), 9.25 (broad s, 1 H, H 6 ).

Photophysical characterization

UV-Visible absorption spectra were recorded on a JASCO V630BIO spectrometer. The steadystate fluorescence emission spectra were obtained by using a JASCO FP8560 spectrofluorimeter instrument. All fluorescence spectra were corrected for instrument response. The fluorescence quantum yields (ΦF) were calculated from equation: ΦF and ΦFR are fluorescence quantum yields of the compound and the reference respectively. A(E) and AR(E) are the absorbance at the excitation wavelength, and n is the refractive index of the medium. IF and IFR are fluorescent intensities of the compound and the reference respectively. 9,10-diphenylanthacene (ΦF = 0.97 in cyclohexane) was used as standard. 35 In all ΦF determinations, correction for the solvent refractive index (η) was applied.

Cell viability assay

The human breast cancer cell line MCF7, human lung cancer cell line A549, human colon cancer cell line HCT116 p53 wild-type (+/+)(obtained from the European Centre of Cell Cultures ECACC, Salisbury, UK) and a p53 null variant of HCT116 (-/-)(kindly provided by Dr. G. Hartleben, University of Groningen), as well as non-cancerous human embryonic kidney cells HEK-293T (provided by Dr. M. P. Rigobello, University of Padova, Italy) were cultured in DMEM (Dulbecco's Modified Eagle Medium).Human ovarian sensitive (A2780) and cisplatin resistant (A2780cisR) cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium. Both media contained GlutaMax supplemented with 10% FBS and 1% penicillin/streptomycin (all from Invitrogen). Cells were incubated at 37°C in a humidified atmosphere of 95% of air and 5% CO2 in an incubator (Heraeus, Germany). In the case of the A2780 and A2780cisR cells, the medium was also supplemented with 2 mM L-glutamine. To maintain resistance, cisplatin-resistant A2780cisR cells were treated with 1 μM cisplatin between every third passage.

For evaluation of toxicity, cells were seeded in 96-well plates (Costar, Integra Biosciences, Cambridge, MA) at a concentration of 10.000 cells/well (A2780, A2780cisR, MCF-7, HEK-293T) or 6000 cells/well (HCT116 p53 +/+, HCT116 p53 -/-, A549) and grown for 24 h in the appropriate medium mentioned above. Solutions of the compounds were prepared by diluting a freshly prepared stock solution (10 -2 M in DMSO for 1 or 10 -3 M inaqueoussolution for cisplatin) of the corresponding compound in media (RPMI or DMEM for the A2780 or A549, MCF-7, HCT116 p53+/+, HCT116 p53-/-and HEK-293T).

Afterwards, 200 L of these dilutions of the compounds were added to the wells to obtain a final concentration ranging from 0 to 120 M, and the cells were incubated for 72 h. Following 24 or 72 h drug exposure, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to the cells at a final concentration of 0.5 mg mL -1 and incubated for 2 h. Then, the culture medium was removed and the violet formazan (artificial chromogenic precipitate of the reduction of tetrazolium salts by dehydrogenases and reductases) dissolved in 200L DMSO. The optical density of each well (96-well plates) was quantified in triplicates at 550 nm using a multi-well plate reader, and the percentage of surviving cells was calculated from the ratio of absorbance of treated to untreated cells. The EC50 was calculated as the concentration reducing the proliferation of the cells by 50% and it is presented as a mean (± SD) of at least three independent experiments.

Statistics

A minimum of three independent experiments were performed with the cells, with four replicates for each condition. The EC50 values were calculated,using GraphPad Prism, as the concentration reducing the viability of the cells or slices by 50%, relative to the untreated samples using a nonlinear fitting of log[compound concentration] versus response and is presented as the mean ± SD of at least three independent experiments.Statistical testing was performed with two-way ANOVA with each individual experiment as random effect. In all graphs and tables the mean values and standard deviation (SD) are shown.

ICP-MS studies

The concentrations of platinum, gold and copper were measured by inductively coupled plasma mass spectrometry (ICP-MS) using an Elan 6000 spectrometer (Perkin Elmer Sciex, Concord, ON, Canada) and Micromist Nebulizer and a cyclonic spraychamber (Glas Expansion Pocasset, MA, U.S.). Nebulizer gas, lens voltage and RF power were optimized daily using a 10 µg/L Pt standard in 0.1% (V/V) HCl and 0.65% (V) HNO3, normal settings were: Rf power 1350 W, nebuliser gas flow rate 0.95 L min -1 , and lens voltgate 10 V. Acquisition parameters were 50 ms dwell time, 1 sweeps reading, 5 replicates and 25 readings per replicate, monitoring all or a subset of 195 Pt + , 196 Pt + , 197 Au + , and 63 Cu + . The instrument was tuned at the beginning of each analysis to ensure optimal operation. Samples were dried by vacuum centrifugation (Eppendorf, concentrator plus) at 60°C for 2 hours. Cell samples were digested using 200 µL 65% HNO3 and 50 µL 30% H2O2 overnight until the solution was clear. Samples were prepared for analysis by dilution to 5 mL with 0.65% (V/V) HNO3 and 0.1% (V/V) HCl. The external standards were prepared from a 10 mg/L Au andPt stock solution (CPI International, Peak Performance, 4400-120213WG01, Lot# 12B214) or Cu stock solution (Plasma CAL, SCL SCIENCE, Q.C. no 4, Cat# 140-102-045, Lot SC5322198) with 0.65% (V/V) HNO3 and 0.1% (V/V) HCl. All reagents were of the highest available purity.

Metal content determination after 24/72h incubation with metal compound +/-inhibitor

A2780 or A2780cisR cells were plated into 25cm 2 culture flasks (CellStar, Grenier Bio, Germany) with RPMI 1640 medium up to a volume of 5 mL per flask. After 24 h, the cells were treated with a mixture of15 µM or 20 µM cisplatin for 24 h, or 5 µM or 10 µM cisplatin for 72 h, +/-cimetidine or +/-CuCl2. Cimetidine was used at 300 µM concentration in H2O,and CuCl2 was used at 30 µM in H2O, both non-toxic to cancer cells. The same set-up was used for compound 1, but the compound was incubated at 5 µM or 10 µM for 24 h, and 3 µM or 6 µM for 72 h, respectively.After 24 hours or 72 hours, the medium was removed, and the cells were washed two times with 3 mL ice-cold PBS to remove dead cells. Thus, lysis buffer (500 µL) was added and the samples were stored on ice for 20 min. Afterwards, cells were detached by using a scrubber policemen and the cell suspension was transferred to a 1.5 mL Eppendorf tube. 200 µL of the sample were transferred to a new Eppendorf tube for ICP-MS analysis, while 5 µL of cell suspension was used for protein determination.

Metal content determination during 120 min incubation with metal compound +/-inhibitor A2780 or A2780cisRcells were plated into 25 cm 2 culture flasks (CellStar, Grenier Bio, Germany) with RPMI 1640 medium up to a volume of 5 mL per flask. The next day, the cells were treated with 5 µM cisplatin or with 3 µM compound 1 (+/-cimetidine or +/-CuCl2) for 10', 20', 30', 60' and 120' min. The samples were processed as described above.

Protein assay

Protein determination was performed using BioRad DC TM Protein Assay reagents A, B and S (Bio-Rad). Using a 96-well-plate, 5 µL of standard (0, 5, 10, 15, 20, 25 and 30 mg/ml bovine serum albumin) and samples were analyzed in duplicate. First, 25.5 µL reagent-mix A+S (25 µL A + 0.5 µL S) was added and then 200 µL reagent B. The absorbance at 610 nm was measured after 15 min (stored in dark) using a PlateReader (Perkin Elmer, EnVision, 2104 Multilabel Reader).

Fluorescence Microscopy

A2780 cells were seeded (5 x 10 5 for each sample) and grown on 8 well microscope plates, coated with Poly-L-lysine hydrobromide (Sigma-Aldrich, P6516) with RPMI medium. After 24h, cells were incubated with various concentrations of Au(III) compound 1 in RPMI, without FCS for 1h at 37 °C. Afterwards, cells were rapidly washed with cold PBS and then fixed with 2% paraformaldehyde for 30 min at 4°C. For visualization of the nuclei with PI (propidium iodide), cells were permeabilized with 0.2% Triton X-100 for 20 min at 4°C and treated with 1 µg/µl of PI for 10 min at room temperature. Cells were washed once with PBS and then analyzed by confocal microscopy. As preparation for visualization, the plate wells were removed from the slide and glycerol was used to cover the slide with a glass cover slip. The fluorescence was analysed using a Leica DM4000 B Automated Upright Microscope, equipped with the appropriate filters. PI was excited at 547 nm (emission wavelength 572 nm) and compound 1 at 358 nm (emission wavelength 461 nm, DAPI filter). The acquired images were obtained using the individual filters and a combined image, overlaying the fluorescence was acquired using the Leica microscope software.

Results

Synthesis and characterization

A novel cyclometallated Au(III) compound 1 was synthesized adapting already established procedures for similar complexes (Figure 2). 26 In details, the C^N precursor [Au(py b -H)Cl2] 34 was reacted with the phosphine3-[4-(diphenylphosphino)phenyl]-7-methoxy-2H-chromen-2-one (PPh2Arcoum) 33 to yield the fluorescent Au(III) compound 1.The complexation of the phosphine ligand with [Au(py b -H)Cl2] as well as the isomeric purity of compound 1 was analyzed by 31 P( 1 H) NMR spectroscopy. TheNMR spectrum (Figure S1a) shows a singlet at 31.5 ppm of the coordinated phosphorous shifted downfield by 35 ppm with respect to the corresponding precursor (Figure S1b). 26 The 1 H NMR spectrum of 1(Figure S2a) shows a downfield shift of the signal of the pyridine proton in position 6 by 0.05 ppm associated with a morphological change of the pyridine moiety. A splitting of the signals corresponding to the carbons of the two phenyl rings compared to the free ligand (Figure S2b) was observed due to their diastereotopic feature induced by the fixed geometry of the cyclometallated ligand as depicted in Figure S2c in the supplementary material. The same splitting of the signals of the two phenyl rings was observed in the 13 C( 1 H) spectrumof 1 (Figure S3).Moreover, another splitting of the signal due to the coupling of the phosphorous atom with carbons and hydrogens is observed.Compound1 in dichloromethane exhibits a typical absorption band at 361 nm and a fluorescence emission band of the coumarin chromophore at 429 nm (Figure S4). As previously observed, the complexation induces a bathochromic shift of the absorption wavelength with respect to free phosphine ligand (+ 33 nm), while there is no significant modification of emission wavelength. 31 It is worth noting that complexation of gold(III) cation preserves the smart character of the coumarinphosphine ligand that we highlighted for gold(I) complexes. 31,32 Indeed, compound 1 displayed a quantum yield of fluorescence almost 8-fold higher than free phosphine (38% and 5% respectively). This phenomenon will make possible to check the stability of the P-Au bond by microscopy of fluorescence experiment in vitro: if the bond breaks, the fluorescence emission will drop dramatically (Figure S5).

Figure2

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Synthesis of compound [Au(py b -H)(PPh2Ar)Cl]PF6 (PPh2Ar = 3-[4 (diphenylphosphino)phenyl]-7-methoxy-2H-chromen-2-one] 1.

Antiproliferative effects

The antiproliferative effects of the gold(III) compound 1 were studied in different human cancer cell lines in comparison to cisplatin, via a classical 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay after 72 h incubation (see experimental for details). Specifically, the selected human cancer cell lines were A2780 and A2780cisR (ovarian carcinoma cisplatin sensitive and resistant, respectively), MCF7 (breast carcinoma), A549 (lung carcinoma) as well as against HCT116 p53 +/+ and -/-(colon carcinoma p53 expressing and knock-out, respectively). The tumour protein p53 is a tumour suppressor gene and known to be downregulated in cisplatin resistant cancer tissues. 36 The obtained EC50 values are summarized in Table 1. Overall, compound 1 exhibits a moderate toxicity profile in all tested cell lines in line with previous studies on similar C^N gold(III) complexes, while being more effective against the ovarian A2780 cancer cells. 26 Compared to cisplatin, 1 shows a moderately higher toxic effect against A2780cisR, which suggeststhat different mechanisms of anticancer activity with respect to the Pt(II) agent may be in place, as well as possible alternative transport and accumulation pathways in this cell line. Instead, no statistically significant difference of EC50 values was observed in the HCT116 p53 -/-cells among the two drugs. This is different from what has been observed for other families of organometallic Au(I) complexes featuring N-heterocyclic carbene ligands. 37 Moreover, similar toxicity with respect to cisplatin was detected in the case of the non-cancerous HEK-293T cells. In addition, we also evaluated the Imax, calculated as the survival rates at maximum inhibition, which provide a measure of drug maximal toxicity, while the EC50 values reflect the drug potency. As it can be seen from the Imax values reported in Table 1, compound 1 shows similar effects as cisplatin, made exception for the HCT116 p53 -/-, MCF7 and A549 cell lines, where the Pt(II) drug has a higher Imax.

Due to the highest activity against the ovarian cancer cells, both sensitive and resistant to cisplatin, these cells were selected for further experiments to investigate the transport mechanisms for the gold(III) compound 1in comparison to cisplatin. Both A2780 and A2780cisR cells were recently evaluated from Sorensen et al. 38 for the expression levels of CTR1, OCT2 and ATP7A/B transporters. Notably, the cisplatin resistant A2780cisR cells show a significantly higher expression of the copper efflux transporters ATP7A (1.3-fold) and ATP7B (5-fold) as well as of the cation uptake transporter OCT2 (1.3-fold). The copper uptake transporter CTR1 is on the other hand less expressed in A2780cisR cells compared to the A2780 (0.4-fold). These data suggest that the resistance observed in the A2780cisR cells is at least partly caused by a different balance between uptake and efflux of cisplatin resulting from a decreased influx via CTR1 and concomitant increased efflux of cisplatin by ATP7A/B, thereby decreasing the intracellular exposure of the cells to cisplatin.

Table 1

Studies on the mechanisms of transport

Competition experiments

Theantiproliferative effectsof compound 1 and cisplatin were determined after 24and 72h incubation in order to observe the development of the toxicity over time (Table 2). Overall, compound 1 is more potent in the A2780 cells, than in the resistant A2780cisR cells at both incubation times. In addition, after 24h incubation, compound 1 is 5-fold more active than cisplatin in A2780 cells, and 7fold more active in A2780cisRcells. However, after 72h incubation, both compoundsdisplay the same cytoxicity in A2780, while the gold(III) compound is still ca. 3-fold more active in A2780cisR than cisplatin. In addition, there is no significant difference between the EC50 values at either 24h or 72h treatment of A2780cisR cells with compound 1. Instead, in A2780 cells, the gold(III) compound shows a 2-fold decrease of the EC50 values from 24h to 72 h incubation, indicating increase ofactivity during chronic exposure.

In order to evaluate the involvement of OCT2 and CTR1 uptake transporters in the metallodrugs accumulation, the compounds' antiproliferative effects were further tested in the presence of transporter inhibitors or competitor substrates, respectively (see experimental section for details). If these transporters are involved in the drug uptake, their inhibition should lead to a reduced intracellular accumulation of the metal complexes and to a decrease in cytotoxic effects, and thus, an increase in EC50 values.Conversely, inhibiting possible efflux transporters such as MATE and ATP7A/B should lead to metallodrug accumulation and enhancement of its antiproliferative effects. Cimetidine (300 µM, Cim) was selected as inhibitor for OCTs and MATE, and CuCl2 (30 µM) as competitive substrate of both CTR1 and ATP7A/B. [START_REF] Spreckelmeyer | [END_REF] Experiments were performed in A2780 and A2780cisR cells after 24and 72h incubation with the compounds. It should be noted that CuCl2 and cimetidine do not show anyanticancer effect on A2780 or A2780cisR cell lines at the selected concentrations (data not shown). The obtained results are summarized in Table 2 and in Figures S6 andS7.

Table 2

After 24h incubation, neither cimetidine nor CuCl2 showed an effect on the potency of gold(III) compound 1 or cisplatin in A2780 cells (Figure S6A). However, in A2780cisR cells (Figure S6B), both cisplatin and compound 1had a significantly increased activity when co-incubated with CuCl2. For cisplatin the resistance was only partly reduced, but for gold(III) compound 1 the resistance was fully compensated resulting in a similar toxicity in both cell lines. These results support the hypothesis that CuCl2may interfere with themetallodrugs de-toxification mechanisms involving the copper efflux transporters (ATP7A/B), which are expressed at much higher levels in the A2780cisR cells than in the wild type A2780 cells. 38 Apparently, these efflux transporters play a minor role in the wild type A2780 cells. Furthermore, the lack of effect of cimetidine on the efficacy of both cisplatin or compound 1(Figure S6A) in both cell lines might indicate that drug uptake processes by OCTs and efflux by MATE are not relevant.

After 72 h incubation of the A2780 cells, both cimetidine and CuCl2 increased the potency of compound 1 significantly, 3.5-fold and 12-fold, respectively. For cisplatin, a 2.5-fold and 4.5-fold increased toxicity could also be observed with cimetidine and CuCl2, respectively (Figure S7A). The same effect was observed in A2780cisR cells, but to a lower extent (FigureS7B). This discrepancy may be rationalized by the hypothesis that for both compounds, at 72 h incubation, efflux mechanisms are inhibited by CuCl2 and cimetidine, possibly via inhibition of ATP7A/B and MATE, respectively. However, it should be noted that, in conditions of chronic drug exposure, other pathways may cause the observed increased cytotoxic effects other than transport mechanisms.

Metal accumulation studies by ICP-MS

After evaluating the toxic effects of both compounds in cancer cells in the presence and absence of transport inhibitors/competitors, the metal content (Au or Pt) was determined by inductively coupled plasma mass spectrometry (ICP-MS) to gain further insights into the drug accumulation mechanisms. For these experiments two concentrations of the metal compounds were chosen based on their EC50values at the selected incubation time (24 or 72 h), namely EC50or 2-fold EC50 concentrations against the A2780 cells were considered for the two drugs.The obtained results are reported in Table 3 and expressed in ng metal/µg protein.

Au accumulation in cancer cells

When cells were treated with compound 1, as expected, the metal content increased as a function of concentration (Table 3 and Figures 3 andS8). Interestingly, no significant differences in Au content could be observed between the two cell lines (sensitive or resistant to cisplatin) treated with the same concentrations of the gold(III) compound 1 at either incubation times. Considering the above mentioned marked differences in EC50 values for 1 between the two cell lines and the different incubation times (Table 2), the differences in accumulation mechanisms may not play a major role in determining the overall antiproliferative effects of the gold(III) complex. Table 3 Figure 3. Au content after 24 h incubation of (A) A2780 (B) A2780cisR cells treated with 5 µM (black) and 10 µM (grey) of compound 1. Pt content after 24 h incubation of (C) A2780 and (D) A2780cisR with 15 µM (black) and 20 µM (grey) cisplatin. Data are expressed as mean ± SD (n = 3).

When cells were co-treated for 24 h with the gold compound and CuCl2, increased accumulation of Au was observed in both cell lines treated with 10 µM concentration of compound 1 (Table 3, Figure 3). The effect of the CuCl2 co-incubation was most marked in the A2780 cells with a 4-fold increase in Au content compared to the controls, whereas the metal content increased only of 2-fold in the A2780cisR. A 2-fold increase was observed at 5 µM in both cell lines, but this increase was not statistically significant. Overall, these results are in line with the enhanced effect of the copper/drug co-incubation on the observed antiproliferative effects (Table 2).

Co-incubation of the gold(III) compound for 24 h with 300 µM cimetidine resulted in a slightly increased accumulation of Au only in the A2780 wild type cells at 10 µM of compound 1, but not at 5 µM, and no effect was seen in the A2780cisR cells (Figure 3) in line with the antiproliferative activity data (Table 2).

The same type of experiments was repeated after 72 h treatment of cells with compound 1 at either 3 or 6 M. After 72 h incubation in the presence of CuCl2, a ca. 2-fold increase in Au content was observed both in A2780 cells and in A2780cisR cells at either metallodrug concentrations, except for the co-incubation of 6 µM 1 in the cisplatin sensitive cells,where no difference was observed with respect to controls (Table 3, Figure S8). This may be explained by the fact that after 72 h, 6 M is about 2-fold the EC50 value for 1 in A2780 cells, which may have caused extensive cell death, altering membrane integrity and Au leakage from the cells. Concerning the cimetidine treatment, this did not have any significant effect on the Au content of the cells after 72 h of incubation (Table 3, Figure S8).

Pt accumulation in cancer cells

Afterwards, the Pt content was evaluated in A2780 and A2780cisR cells treated with cisplatin after 24(Table 3, Figure3) or 72h incubation (Table 3, Figure S9) by ICP-MS. At 24 h, the Pt content was the same for both cell lines, despite the known differences in the expression of hCTR1 and OCT transporters. 38 The increase in metal content was concentration dependent over 24 h in the A2780 cells, while a similar trend could not be observed in the cisplatin resistant cells, where the Pt content remained substantially unaltered at both drug concentrationsand incubation times (Figure 3 andS9).This concentration dependent effect could not be observed in the case of the A2780cisR cells also after 72 h incubation, supporting the idea that higher detoxification and possibly efflux mechanisms may be in place. Notably, no significant difference in Pt content could be observed after co-incubation with either cimetidine or CuCl2. Thus, these data cannot explain the observed increase in anticancer effects (Table 2).

Copper accumulation in cancer cells

The viability results of cisplatin treated human ovarian cancer cells showed a decreased EC50 value after co-incubation with CuCl2. However, the ICP-MS data did not show an increased Pt content that could be responsible for such an effect. In order to reinforce the evidence that inhibition of the copper transporters by cisplatin or by the Au(III) complex is not relevant to their mechanism of anticancer effects, we investigated whether the Cu 2+ content was increased due to the co-incubation with each metallodrug. Concerning copper uptake, CTR1 affinity to Cu is between 0.6 µM in fibroblasts and 13 µM in murine hepatocytes, 39 whileATP7B binding affinity to Cu is 2.5 * 10 -17 M. 40 Both transporters are not only selective for Cu, but also for example for Ag(I), Cd(II) and Fe(III). 41 Initially, copper content was evaluated in both the wild-type A2780 and cisplatin resistant A2780cisR cells by ICP-MS since the different CTR1/ATP7A/B transporter expression levels 38 may have an effect in its accumulation. The obtained results show that both cell lines have the same basal copper content (0.056 ± 0.02 ng Cu in A2780 and 0.045 ± 0.01 ng Cu in A2780cisR). Furthermore, treatment with CuCl2 (30 µM) did not significantly alter these basal levels (Figure 4).

Concerning metallodrug treated cells, it could be observed that the copper content did not decrease in the cell lines upon treatment with the Au(III) compound 1 for 24 (5 and 10 µM) and 72 h (3 and 6 µM), at any selected drug concentration (Figure 4). Instead, co-incubation of 1 with CuCl2 induced an increased copper accumulation compared to controls after 24 h in both cell lines. At 72 h, the Cu content was markedly increased mainly in the A2780 cells. Overall, the ICP-MS data showed an increased Au and Cu content after 24 h and 72 h incubation in the CuCl2 co-incubation experiments, which may be responsible for the observed increased antiproliferative effects in the same cells (Table 3). The effect of the increased Cu-content is not fully understood yet, but an additive or even synergic anticancer effect with Au may be possible.

Considering cisplatin treatment, no significant in the Cu content between the controls and cisplatin treated cells could be observed in both cell lines after 24 and 72 h incubation (Figure 5). Instead, after 24 h, the Cu content significantly increases at both concentrations of cisplatin coincubated with CuCl2 in both cell lines. However, this effect was absent in the A2780cisR cells incubated for 72 h. The ICP-MS data showed no increased Pt and Cu content after 24 h and 72 h incubation in the CuCl2 co-incubation experiments (Table 3). To summarize the results concerning the gold(III) complex, the following can be concluded: after 24 h, co-incubation of 1 with CuCl2, but not with cimetidine, induced a significantly higher antiproliferative effects in A2780cisR cells, whereas in A2780 cells no effect was seen of either CuCl2 or cimetidine. After 72 h incubation, co-incubation of 1 with CuCl2 resulted in an increase in cytotoxicity and increase in Au and Cu content in both cell lines as demonstrated by ICP-MS. Based on these results two explanations can be suggested for these effects: i) compound 1 is a substrate for ATP7A/B, and inhibition of the efflux transporter ATP7A/B by excess CuCl2 results in a higher Au accumulation and higher drug potency, and/or ii) the increased overall cytotoxicity is a result of additive or synergistic effects of Au and Cu accumulation via different transport mechanisms. Furthermore, a direct involvement of the CTR1 or OCT2 in the uptake of the gold drug, as well as of MATE in its efflux, could not be supported based on our studies. The increased efficacy of compound 1 in the presence of cimetidine after 72 h of incubation in A2780 cells, but not after 24 h, is difficult to explain, as no concomitant increased accumulation of Au was observed by ICP-MS. Studies with other, more selective, inhibitors for each single drug transporter are needed to confirm these results.

Concerning cisplatin, similarly to compound 1, 24 h drug incubation with CuCl2 shows a significant decrease in viability in A2780cisR cells, although not as strong as for the gold(III) compound 1. After 72 h, co-incubation of cisplatin with either cimetidine or CuCl2 leads to an increase in cytotoxicity, but no increase in Pt content could be observed in both cell lines as demonstrated by ICP-MS. This result, together with the evidence for increased Cu content in A2780 cells, leads to the hypothesis that copper accumulation is the reason for the observed enhanced anticancer effects in this cell line.

Metal uptake studies

In order to specifically evaluate uptake mechanisms of metallodrugs in cancer cells, we measured the metal content (Au, Pt) in both A2780 and A2780cisR at different time points during the first 120 min of incubation in cells by ICP-MS. Thus, samples were taken after 10, 20, 30, 60 and 120 min and the obtained results are reported in (Figures6 and 7).

In the case of compound 1, co-treatment with cimetidine did not affect the Au uptake in both cell lines at any recorded concentration. When cells were co-incubated withCuCl2, a significantly higher Au content could be observed only after 120 min in the A2780 cells (Figure 6A), but not in the cisplatin resistant ones (Figure 6B). Overall, both cimetidine and CuCl2 do not change the uptake of compound 1. It should be noted that, between 10-20 min incubation, a drop in Au content can be observed in A2780 cells. We do not have an explanation for that thus far, but it should be further investigated in additional experiments.

In the case of cisplatin, the Pt content in cells co-incubated with cimetidine or CuCl2 did not show any difference over 120 min in both cell lines (Figure 7). A similar trend was observed in the co-treatment with cimetidine, made exception for the significantly increased Pt content recorded at 120 min in A2780 cells.Considering the whole set of data, it is also worth noting that metal accumulation is more efficient in the case of the gold(III)compound 1 compared to cisplatin in both cancer cell lines over time (see summary of the obtained data in Table 3). In line with the aforementioned accumulation studies, a direct involvement of OCTs or CTR1 in the uptake of cisplatin was not observed. Otherwise, inhibition by cimetidine should have resulted in lower cytotoxicity and lower Pt, accumulation in the cells, which instead was not recorded. It should be also considered that Holzer and Howell showed that, as a consequence of cisplatin and copper incubation, the CTR1 transporter undergoes rapid down-regulation in human ovarian 2008 cancer cells, and that it might be internalized from the membrane, potentially leading to a decreased cisplatin uptake. 13 However, a decreased Pt uptake was also never evidenced in our results. Similarly, a direct involvement of MATE or ATP7A/B in the accumulation of cisplatin could not be confirmed.

Fluorescence microscopy studies

To investigate the sub-cellular localization of the Au complex, we performed fluorescence microscopy experiments on A2780 cells treated with Au(III) compound 1 and the two inhibitors cimetidine and CuCl2. Propidium iodide (PI) staining was used to evidence cell nuclei. The pictures show the A2780 cells after 2h incubation with gold(III) compound 1(2-fold the EC50) (Figure 8). The strong fluorescence observed indicates that the coumarin-phosphine ligand is still coordinated to gold(III) cation. The compound itself does not co-localize with PI in the nuclei, but clearly enters the cells and is localized in organelles in the cytoplasm. For examples, a similar distribution pattern was observed for a gold(I) complex featuring the same coumarin ligand as 1. 32 In this case, the gold(I) complex was hypothesized to accumulate in lipid rafts, cellular domains containing plasma membrane proteins and lipids, which areinvolved in the regulation of numerous cell functions, including signalling, trafficking, migration, adhesion, and growth. This observation points towards a different mechanism of action compared to cisplatin and suggests that the DNA damage may not play a pivotal role in the toxicity of the Au compound. Co-incubation with either cimetidine or CuCl2 did not change the intracellular accumulation pattern (Figure 8).

Conclusions

In the past years, several studies have been carried out to elucidate the mechanisms of uptake and efflux of cisplatin in cancer cells and kidney slices, as well as in numerous in vitro models of cancer cells. [START_REF] Spreckelmeyer | [END_REF] Despite the numerous research efforts, such transport mechanisms are not yet fully understood. In addition, the mechanisms leading to activity/toxicity and cellular accumulation of new experimental anticancer gold complexes have received scarce attention,albeit the numerous compounds' structures published in the literature. Recently, we have reported on a promising family of gold(III) C^N complexes as cytotoxic agents, with [Au(py b -H)PTACl] (py b -H = C^N cyclometallated 2-benzylpyridine) 26 (Figure 1) as one of the most effective derivatives in cancer cells in vitro. In order to broaden the scope of our work and develop a compound trackable in cells via fluorescence microscopy, we report here on the cytotoxic effects of a newfluorescent gold(III) C^N complex1studied in a panel of human cancer cell lines in comparison to cisplatinin vitro.The results show that 1 is more active than cisplatin in a number of cell lines, including MCF7, HCT16 p53 -/-, and A2780cisR cells. However, it has similar antiproliferative effects for other cancer cells, including the ovarian A2780 cells. The compound is also moderately cytotoxic towards the non-tumorigenic HEK cells. Additionally, we evaluated for the first time the involvement of different uptake and efflux transportersnamely OCTs, CTR1, MATEs, and ATP7A/Bin the accumulation mechanisms of 1in human ovarian cancer cells in vitro, using competitive inhibitors/substrates and determining the intracellular metal content by ICP-MS.The results are compared with those obtained for cisplatin in the same experiments.

The uptake and intracellular accumulation of the gold(III) compound 1 is more efficient than that of cisplatin, as judged from the metal content assessed in both A2780 and A2780cisR cells by ICP-MS. In general, while uptake transporter OCT2 and CTR1 are unlikely to be involved in the transport mechanisms of both compounds in the selected cell lines, as well as in their antiproliferative effects, the efflux transporters ATP7A/B should be investigated further in the case of the gold complex. The preliminary fluorescence microscopy studies indicate that the gold compound accumulates intracellularly mainly in cytoplasmic organelles other than nuclei. Further studies are necessary to establish if these corresponds to mitochondria or lysosomes among others.

Overall, these initial results may constitute the basis for future more extensive studies on experimental metallodrugs transports and their relevance to the mechanism of activity/resistance towards different types of cancer, highlighting the differences with classical platinum-based chemotherapeutics.

Table 2. EC50 values of compound 1 and cisplatin against A2780 and A2780cisR cells, incubated in absence and presence of CuCl2 or cimetidine (Cim), recorded after 24 h and 72 h incubation. The reported values are the mean ± SD of three independent experiments. For statistical analysis, the two way ANOVA was used. * (p ≤ 0.05), ** (p ≤ 0.01) indicate the difference is significant when compared to samples treated with the metallodrugs only (control).

Figure 1 .

 1 Figure 1. The anticancer Pt(II) drug, cisplatin, and cyclometallated [Au(py b -H)PTACl] (py b -H = C^N cyclometallated 2-benzylpyridine, PTA = 1,3,5-triazaphosphaadamantane).

13 C( 1 H

 131 ) NMR (Acetone-d6, 125.77 MHz, 300 K): 47.9 (s, CH2-PyrBz), 56.5 (s, O -CH3), 101.2 (s, CH D ), 113.8 (s, CH C ), 114.0 (s, Cquat-coum), 122.9 (s, Cquat-p-C6H4), 124.0 (d, 1 JP-C = 84.3 Hz, Cipso-Ph), 124.5 (d, 1 JP-C = 83.0 Hz, Cipso-Ph), 124.8 (d, 1 JP-C = 70.4 Hz, Cipso-p-C6H4), 125.4 (d, 4 JP-C = 3.8 Hz, CH 5 ), 127.3 (d, 4 JP-C = 3.8 Hz, CH 3 ), 128.8 (s, CH 4' ),128.9 (d, 4 JP-C = 2.5 Hz, CH 5' ),129.8 (s, CHpara-Ph), 129.9 (s, CH 3' ), 130.2 (d, 2 JP-C = 10.1 Hz, CHortho-Ph), 130.3 (d, 2 JP-C = 10.1 Hz, CHortho-Ph), 131.0 (s, CH B ), 133.7 (d, 3 JP-C = 7.5 Hz, CH 6' ), 134.5 (d, 3 JP-C = 2.5 Hz, CHortho-p-C6H4), 134.6 (s + d, 3 JP-C = 2.5 Hz, Cquat-Bz + CHortho-p-C6H4), 136.

Figure 4 .

 4 Figure 4. Evaluation of copper content in human ovarian cancer cellstreated with 1. Cu content in A2780 (WT) and A2780cisR (Res) cells after (A) 24 h and (B) 72 h incubation with 30 µM CuCl2 and different concentrations of compound 1. Data are expressed as mean ± SD (n=3).

Figure 5 .

 5 Figure 5. Evaluation of copper content in human ovarian cancer cellstreated with cisplatin. Cu content in A2780 (WT) and A2780cisR (Res) cells after (A) 24 h and (B) 72 h incubation with 30 µM CuCl2 and different concentrations of cisplatin. Data are expressed as mean ± SD (n=3).

Figure 6 .

 6 Figure 6. Gold uptake determination in cancer cells -Au content [ng metal/µg protein] in A2780 (A) and A2780cisR cells (B) treated with 1 and co-treatment with cimetidine or CuCl2. Data are expressed as mean ± SD (n=3).

Figure 7 .

 7 Figure 7. Platinum uptake determination in cancer cells -Pt content [ng metal/µg protein] in A2780 (A) and A2780cisR cells (B) treated with cisplatinand co-treatment with cimetidine or CuCl2. Data are expressed as mean ± SD (n=3).

Figure 8 .

 8 Figure 8. Fluorescence microscopy of A2780 cells treated for 2 h with compound 1 (5 µM) with/without 300 µM cimetidine or with/without 30 µM CuCl2.

Acknowledgements

Authors thank Cardiff University for funding. The University of Groningen and the University of British Columbia are acknowledged for cofounding the PhD fellowship of S.S.. Prof. G.M.M. Groothuis and Dr. I.A.M. de Graaf are kindly acknowledged for help in the interpretation of the data. Prof. I. H. Lambert is kindly acknowledged for providing aliquots of the A2780 and A2780cisR cancer cells.E.B. and B.B. thank the Conseil Régional de Bourgogne Franche-Comté, the Ministère de l'Enseignement Supérieur et de la Recherche, the Centre National de la Recherche Scientifique (CNRS), the French Research National Agency (ANR) via project JCJC "SPID" ANR-16-CE07-0020for financial support. This work is part of the project PHARMACOIMAGERIE ET AGENTS THERANOSTIQUES and of the project CHIMIE DURABLE, ENVIRONNEMENT ET AGROALIMENTAIRE, supported by the Université de Bourgogne, Conseil Régional de Bourgogne through the plan d'actions régional pour l'innovation (PARI) and the European Union through the PO FEDER-FSE Bourgogne 2014/2020 programs.

Data Availability Statement

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Author Contributions

AC, SS, SS contributed conception and design of the study. EB, BB and AC contributed to the design, synthesisand characterization of the gold compound. SS performed the anticancer studies in vitro and the data interpretation. SS and MVDZ performed the ICP MS experiments and the statistical analysis. AC and SS wrote the first draft of the manuscript, while SS, EB and BB wrote sections of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.

All relevant data is contained within the manuscript:All datasets [GENERATED/ANALYZED] for this study are included in the manuscript and the supplementary files.


Clara Letierce

Colleen Mills

Nicolas Arnaud

Empowering middle managers to free their strategic capabilities Journal of Organizational Change Management (

Keywords: organizational change, middle managers, empowerment, change translation, strategizing

Empowering middle managers to free their strategic capabilities

INTRODUCTION

Current new ways of organizing have underlined the disadvantages of a tall hierarchy, arguing that middle managers impede flexibility whereas flat organizations empower employees, enhance reactivity and performance [START_REF] Carney | Freedom, Inc: How Corporate Liberation Unleashes Employee Potential and Business Performance[END_REF][START_REF] Hamel | First, let's fire all the managers[END_REF][START_REF] Lee | Self-managing organizations: exploring the limits of lesshierarchical organizing[END_REF]. In contrast, other scholars have emphasized middle managers' strategic role, especially during organizational change [START_REF] Arnaud | Materialising strategy in mundane tools: The key to coupling global strategy and local strategy practice[END_REF][START_REF] Sukoco | Middle manager capabilities and organisational performance: the mediating effect of organisational capacity for change[END_REF], and suggested that their leadership is often prevented by timeconsuming administrative activities [START_REF] Detchessahar | Quels modes d'intervention pour soutenir la discussion sur le travail dans les organisations ? @[END_REF][START_REF] Kieran | Strategic change and sensemaking practice: enabling the role of the middle manager[END_REF]. These ambivalent perceptions of middle managers are problematic because they blur our understanding firstly of their value in an organization [START_REF] Hamel | Excess management is costing the U.S. $3 trillion per year[END_REF][START_REF] Galière | When food-delivery platform workers consent to algorithmic management: a Foucauldian perspective[END_REF], and secondly of the conditions that could enable to release their strategic capabilities [START_REF] Gomez | The leader-member exchange as a link between managerial trust and employee empowerment[END_REF].

These insights motivated us to conduct a qualitative case study in a French bank (FB) that decided to fly in the face of contemporary thinking and provide middle managers with more time and resources to engage in change translation. Previous research has already emphasized the importance of empowering this 'interface' between top-management and front-line employees [START_REF] Buick | Enabling Middle Managers as Change Agents: Why Organisational Support Needs to Change[END_REF][START_REF] Raelin | Whither Middle Management? Empowering Interface and the Failure of Organizational Change[END_REF], and we aim to go further by asking the following question : How empowered middle manager engage in change translation?

We choose to rely on 'strategy as practice' as a theoretical framework [START_REF] Johnson | Micro strategy and strategizing : towards an activity-based view[END_REF], which renewed concerns about the doing of strategy [START_REF] Jarzabkowski | If you aren't talking about practices, don't call it a practice-based view : Rejoinder to Bromiley and Rau in Strategic Organization[END_REF]. More precisely, we will build on the notions of building and dwelling strategizing proposed by Chia and Rasche (2015), to analyze the micro-practices of empowered middle managers during organizational change.

By doing so, this article aims to provide three major contributions. We first seek to enrich the strategy as practice stream of research by analyzing how building and dwelling strategic practices can be associated. By studying an original empirical context, we also aim to better understand the role of 'disempeded' middle managers during organizational change translation. Finally, this study allows to give practical insights on the conditions needed to release managers from managerialism and develop empowering leadership.

This paper begins by reviewing the ambivalent perceptions of middle managers in our contemporary organizations before presenting the 'strategy as practice' analytical grid, and the notions of building and dwelling strategizing. We then describe the context of our original case setting and the research design. The findings will follow, and lead to both theoretical and practical contributions.

LITERATURE REVIEW Middle managers perceived as obstacles to organizational efficiency

Many new ways of organizing such as sociocracy [START_REF] Endenburg | Sociocracy: The organization of decision-making[END_REF][START_REF] Romme | Management as a science-based profession: a grand societal challenge[END_REF], Holacracy [START_REF] Kumar | Holacracy-the future of organizing? The case of Zappos[END_REF][START_REF] Robertson | Holacracy: The revolutionary management system that Abolishes hierarchy[END_REF] or 'liberated organizations' [START_REF] Getz | Liberté & Cie[END_REF][START_REF] Peters | L'entreprise libérée[END_REF][START_REF] Sferrazzo | Are liberated companies a concrete application of sen's capability approach[END_REF] are questioning hierarchical authority and encourage the redistribution of decision-making power from managers to frontline workers [START_REF] Chen | The mediating role of decentralization between strategy and performance: Evidence from Danish firms[END_REF]. In these initiatives, management is seen as something that concerns everyone, and everybody becomes a manager. Employees' tasks are enriched with managerial attributions such as planification, resource repartition or control, and middle managers thus become potentially redundant [START_REF] Hermkens | The Role of Middle Management in Continuous Improvement: The Bermuda Triangle of Leadership, Implementation and Behavioral Change[END_REF].

Three main arguments support this criticism against middle managers. Firstly, middle managers are costly as they are often qualified or experienced workers, and their added value is difficult to measure [START_REF] Hamel | Excess management is costing the U.S. $3 trillion per year[END_REF][START_REF] Galière | When food-delivery platform workers consent to algorithmic management: a Foucauldian perspective[END_REF]. Secondly, middle managers are intermediaries in the decision-making line. Their presence makes thus the organization less flexible and increasingly disconnected from the real problems experienced by frontline workers [START_REF] Gittell | Supervisory span, relational coordination and flight departure performance: a reassessment of postbureaucracy theory[END_REF][START_REF] Handel | Theories of lean management : An empirical evaluation[END_REF]. These arguments foster the idea that middle managers are obsolete 'dinosaurs' [START_REF] Floyd | Dinosaurs or dynamos? Recognizing middle management's strategic role[END_REF][START_REF] Holmemo | Bypassing the dinosaurs? How middle managers become the missing link in lean implementation[END_REF], all the more in a particularly unstable and turbulent environment. Thus, to be more responsive and agile, many organizations are motivated to flatten their structure [START_REF] Bodrožić | The Evolution of Management Models: A Neo-Schumpeterian Theory[END_REF][START_REF] Lee | Self-managing organizations: exploring the limits of lesshierarchical organizing[END_REF], by reengineering traditional hierarchies to create functional teams in order to eliminate management layers [START_REF] Van De Kamp | Holacracy-A radical approach to organizational design[END_REF]. Finally, literature on front-line employees' empowerment suggests that many practices such as job enlargement and enrichment (Hut & Molleman, 1998) or delegating responsibilities [START_REF] Kirkman | Beyond self-management: antecedents and consequences of team empowerment[END_REF] have significant implications for organizational effectiveness [START_REF] Rauch | A meta-analysis of different HR-enhancing practices and performance of small and medium sized firms[END_REF]. These studies highlight the utility of giving teams more autonomy and suggest that a reconsideration of middle managers' contribution is needed. Such thinking give weight to calls to flatten hierarchies by reducing the number of middle managers in order to increase profitability: "the middle-manager role has been seen as one to eliminate.

Pressures on organizations in the increasingly global marketplace to be more cost conscious, adaptable and flexible have made middle managers a target because they are seen to add costs, slow down decisions and obstruct the flow of information" (Balogun, 2003, p. 70).

Middle managers seen as impeded strategists

Alternatively, a growing body of research relying on [START_REF] Mintzberg | The nature of managerial work[END_REF][START_REF] Mintzberg | Manager's job: folklore and facts[END_REF] and [START_REF] Floyd | Middle management involvement in strategy and its association with strategic type: a research note[END_REF] seminal works, has sought to underline the substantial role of middle managers during strategic change [START_REF] Balogun | Organizational restructuring and middle manager sensemaking[END_REF][START_REF] Balogun | Strategy-as-practice research on middle managers and sensemaking[END_REF][START_REF] Heyden | Rethinking 'top-down'' and "bottom-up'' roles of top and middle managers in organizational change: Implications for employee support[END_REF][START_REF] Mckenzie | Energizing middle managers' practice in organizational learning[END_REF][START_REF] Teulier | Middle managers' sensemaking and interorganizational change initiation: translation spaces and editing practices[END_REF][START_REF] Van Der Voet | Change Management in Hard Times: Can Change Management Mitigate the Negative Relationship Between Cutbacks and the Organizational Commitment and Work Engagement of Public Sector Employees[END_REF]. For example, [START_REF] Rouleau | Middle managers, strategic sensemaking, and discursive competence[END_REF] highlighted how middle managers, through their daily activities, interpret and "sell" strategic changes. Similarly, Arnaud, Mills and Legrand (2016) studied how middle managers participate in the process of worker liberation through discursive practices that contribute to the collective dynamics of sensemaking and sensegiving. It seems, middle managers' unique position enables them to facilitate change by helping employees to give meaning to it, and thus reduce resistance to change [START_REF] Buick | Enabling Middle Managers as Change Agents: Why Organisational Support Needs to Change[END_REF][START_REF] Johannsdottir | Leadership role and employee acceptance of change: Implementing environmental sustainability strategies within Nordic insurance companies[END_REF][START_REF] Radaelli | Middle managers and the translation of new ideas in organizations: a review of micro-practices and contingencies[END_REF].

To pursue these thoughts, some studies have suggested that middle managers' lack of constructive engagement pointed out by some, might be explained not by their obsolescence, but because they are caught up in time-consuming duties, such as reporting and meetings, in order to meet the requirements of command-and-control bureaucracies [START_REF] Denning | The reinvention of management[END_REF][START_REF] Walker | Command and control: the sociotechnical perspective[END_REF]. The consequence is that middle managers have less time to serve frontline activities and are impeded in developing and applying their strategic capabilities [START_REF] Detchessahar | Faire face aux risques pshycho-sociaux: quelques éléments d'un management par la discussion[END_REF]. While some may consider this managerial absence as 'liberatory' [START_REF] Carney | Freedom, Inc: How Corporate Liberation Unleashes Employee Potential and Business Performance[END_REF][START_REF] Hamel | First, let's fire all the managers[END_REF], these authors argue that such situations foster discomfort and disengagement among not only middle managers but also their subordinates who miss their support [START_REF] Detchessahar | Quels modes d'intervention pour soutenir la discussion sur le travail dans les organisations ? @[END_REF]. Thus, the main challenge is to support managers' to develop their change agent capabilities by giving them more time, resources and valuing their contribution to organizational change translation [START_REF] Buick | Enabling Middle Managers as Change Agents: Why Organisational Support Needs to Change[END_REF][START_REF] Detchessahar | Santé au travail. Quand le management n'est pas le problème, mais la solution[END_REF][START_REF] Kieran | Strategic change and sensemaking practice: enabling the role of the middle manager[END_REF].

Strategic practices through a micro lens perspective

The purpose of this article is to analyze how empowered middle managers contribute to organizational change translation in practice. To do this, we choose to be guided by the 'practice turn' [START_REF] Nicolini | The practice approach: For a praxeology of organisational and management studies[END_REF][START_REF] Schatzki | The practice turn in social theory[END_REF]. This approach introduced the 'strategy as practice' (SaP) conceptual framework which shifted the focus from strategy content to the micro strategic work [START_REF] Jarzabkowski | Constructing spaces for strategic work: a multi-modal perspective[END_REF][START_REF] Whittington | Completing the practice turn in strategy research[END_REF][START_REF] Whittington | Strategy as practice, process, and institution: Turning towards activity[END_REF].

This new conception of 'strategizing' had several implications in strategy analysis (cf. the special issue of British Journal of Management, 26, 2015;[START_REF] Chia | Post-processual challenges for the emerging strategy-as-practice perspective: discovering strategy in the logic of practice[END_REF][START_REF] Johnson | Micro strategy and strategizing : towards an activity-based view[END_REF][START_REF] Miettinen | Re-turn to practice : an introductory essay[END_REF]. First, it encourages researchers to focus their attention on practitioners' praxistheir sayings and doings as they enact their company strategy [START_REF] Johnson | Micro strategy and strategizing : towards an activity-based view[END_REF]. Second, it paves the way for all organizational members including middle managers to be recognized as strategy practitioners (Arnaud, Mills, Legrand, et al., 2016;[START_REF] Rouleau | Micro-Practices of Strategic Sensemaking and Sensegiving: How Middle Managers Interpret and Sell Change Every Day[END_REF][START_REF] Van Rensburg | Making strategy work: The role of the middle manager[END_REF]. Third, it introduced the idea that strategizing was not only elaborated with planned decisions but could also be conceived as an articulation of both planned and emergent practices [START_REF] Chia | Strategy as practical coping: a Heideggerian perspective[END_REF][START_REF] Tsoukas | On organizational becoming: rethinking organizational change[END_REF], that could lead to unintended outcomes [START_REF] Balogun | From intended strategies to unintended outcomes: The impact of change recipient sensemaking[END_REF].

These reflections were then pursued by more recent work of Chia and Rasche (2015) who proposed the notions of building and dwelling visions of strategizing [START_REF] Chia | Strategy-as-practice: reflections on the research agenda[END_REF][START_REF] Chia | Strategy as practical coping: a Heideggerian perspective[END_REF][START_REF] Chia | Post-processual challenges for the emerging strategy-as-practice perspective: discovering strategy in the logic of practice[END_REF][START_REF] Golsorkhi | Cambridge handbook of strategy as practice[END_REF][START_REF] Rasche | Researching Strategy Practices: A Genealogical Social Theory Perspective[END_REF]. By doing so, the authors encourage an extension in the understanding of strategy emergence process: "what is needed […] is a redirection of attention from the declared overt activities traditionally associated with strategizing to the subtle maneuvers adopted by individuals, organizations and businesses over the course of dealing with pressing immediate concerns that threaten their survival, growth and development" p.12). Chia & Rasche, 2015, p.7) The authors thus suggest that practice researchers should revise their understanding of the emergence process of strategy, giving better attention to "the myriad, micro activities that make up strategy and strategising in practice" [START_REF] Johnson | Micro strategy and strategizing : towards an activity-based view[END_REF]. Dwelling and building perspectives of strategizing, however, should not be understood as independent. On the contrary, they should be treated as complementary perspectives necessary for fully analyzing strategy emergence.

Table 1. Comparison of building and dwelling epistemology (adapted from

Relying on this theoretical framework, we propose to examine both planned and spontaneous middle managers' strategizing. To do this, we captured both 'purposeful' practices guided by top management's strategic instructions and 'purposively' undertaken practices that emerged as workers dealt with the obstacles that reality presents. In both cases, the practices observed are considered as strategic for two main reasons. First, they deal with organizational change implementation, and more precisely to organizational restructuring [START_REF] Balogun | The Practice of Organizational Restructuring:. From Design to Reality[END_REF]. Second, they

Building mode Dwelling mode

Strategizing is conducted in a distinct and detached space from the situation they are in.

Strategizing is intimately immersed and inextricably intertwined with the local context.

Actions are guided by predefined goals directing efforts towards outcomespurposeful action.

Actions are directed towards overcoming immediate impedimentpurposive practical coping.

Consistency of action assumed to be ordered by deliberate intent defined by topmanagement strategy.

Consistency of action assumed to be ordered by an internalized disposition of topmanagement strategy.

were enacted in order to cope with local issues in 'the here and now'. As Chia and Rasche (2015) note, "attending to and dealing with the problems, obstacles and concerns confronted in the here and now may actually generate a surprising consistency of action that through hindsight may appear as a relatively stable pattern to which we might ascribe the label 'strategy'" (Chia & Rasche, 2015, p.12).

METHODS

As stated previously, the aim of this study is to analyze middle managers' organizational change translation micropratices. To do so, we opted for a qualitative approach that enable to observe and gather rich testimonies in two distinct banking branches belonging to a large French bank (FB).

The two units of this embedded case study (Yin, 2014) were chosen for both their similarity and their distinctiveness. While both were located in urban areas, their size differed because one unit was composed of three branches while the other had four. This difference was significant as we reasoned that size could influence managers' availability for strategic activity. Before describing our methodological design, we will present the empirical context of this study.

The case: a bank's re-engineering project

Facing considerable transformations in technology, political regulations in retail banking and global competition [START_REF] Łasak | Transforming the scope of the bank through fintechs: Toward a modularized network governance[END_REF], FB decided to reconfigure its organizational structure to offer a better service to customers and increase its productivity.

FB's first objective was to optimize the regional network grouping branches into larger entities, what we refer to as 'units' (see Figure 1), to avoid the understaffing and overworking problems, and thus to optimally meet customers' real needs. Consequently, an updated governance manages the units: they are now led by a management team rather than a single branch manager. In practice, each unit came to be managed by a Branch Manager (BM) who manages the Business Account Managers (Bus AM) and is the line manager of the other two members of the management team;

an Assistant Branch Manager (ABM) who is therefore responsible for Personal Account Managers (Pers AM) in charge of the retail market, and an Operations Manager (OPM) in charge of regulatory and quality aspects. This OPM is also responsible for receptionists (RECs). Figure 1 This new structure is designed to empower managers through different means. For example, middle managers are freed from direct customer accounts management so they can be dedicated to their managerial tasks. Also, with the implementation of the managerial team, the responsibilities are shared and each of them can focus on their team's expertise to better respond to their specific needs.

This new structure also gives them the opportunity to discuss between middle managers, to share their difficulties and brainstorm on possible solutions.

independently of top management surveillance. These precautions were necessary to take into account the ethical issues of this research and to obtain the consent of the respondents [START_REF] Brinkmann | Ethics in qualitative psychological research[END_REF].

Finally, we conducted 28 semi-structured interviews with employees and managers from the two banking branches studied (see Appendix 1). These interviews were designed to discuss the managers' translation practices observed, and to identify new ones with questions such as 'what were, in your opinion, the main transformations following this project? How do they take shape in your daily work?', 'Do you know how the other banking branches are implementing this new way of working?', 'was this practice explicitly asked by the top management or written in the project booklet?', etc. The interview guide was used as a support for the exchange with the interviewees, but the order of the questions varied according to the interviews in order to keep the flow of the discussion fluid.

FINDINGS

The findings present managers' organizational change translation micropratices when released from high levels of managerialism. The three following sections are structured depending on the outcome of manager's strategizing and distinguish building and dwelling practices.

Federate the team spirit to facilitate collaboration

One of the main challenges of the new organization structure was to enhance a sense of solidarity across the banking branches of one unit.

According to the prescriptions in the change booklet, both banking branches organized paired working between the banking branches of a unit. However, we observed dwelling strategizing in the way the pairs were conceived. In the first unit, the manager made duos within the same banking branch, while in the second the duos were created with members drawn from different banking branches of their unit:

"I don't think that it is in the [booklet] but the will was really to have a continuity in the response to our customers, it was the first objective I associated exchanges between sites and mobility between sites, that's why I actually made these pairs between sites" (BM#2).

Another dwelling strategizing practice concerned commercial challenges. As the following excerpts explains, the management team decided to create teams across the bank branches' activities. This meant that every team was composed of three people: one personal account managers, one business account manager and a receptionist:

"we made three teams which mixed the people from all the branches and all the markets, including the people from the reception. […] As the leader, I used to send an email everyday with the daily results and I was supporting my team, giving advices or showing how to do if they needed to, because it may not have been their priority before" (Pers AM#4)

"Being mixed up between professions is good because I necessarily produce less than a particular account manager […]. I think it's really nice to have mixed us by profession but also by agency because it allows us to be interested in other structures and to have interactions. I think that this

is essential because otherwise it would put in opposition the agencies and then instead of creating emulation, it would create discord". (Bus AM#7)

As mentioned by the employees above, this idea developed the solidarity and interdependence between the various professions. It also encouraged the collaboration between the banking branches and a combined sense of team spirit.

In the same perspective, we noted during our observations that logistic issues were also taken into consideration by managers. Indeed, one manager decided to alternate meetings' location between the different banking branches of his unit and made sure that every employee could attend by extending the duration of branch closures:

"my manager said 'the meetings won't be all at [banking branch 1], so we have turns. He considers us equally, there is not the big agency and us, we are one unit" (Bus AM#1).

"Thursday meeting was supposed to happen from 1:45pm to 3pm, and we asked an exemption to extend the duration until 3:30pm for everyone go back in terms of journeys, it was too short and it creates useless stress" (ABM#2). This is a clear example of a dwelling micro-strategizing designed to enhance inclusivity while advancing the objective of creating a sense of unity across diverse banking groups.

Develop employees' capabilities to improve customer satisfaction

The second main objective of the organization project was to improve customer satisfaction by developing employees' capabilities.

In line with top-management's prescription, we observed that both banking branches organized sales meetings that separated the retail market, business markets and receptionists. The meeting frequency proposed by top-management was also respected (i.e., once a week for the retail market and once every two weeks for the business market and the receptionists).

"This morning I animated my first business meeting with a specific part on the complete strategy that we are going to apply in their market with a focus on how their activities contribute to the banking branch results" (BM#1)

"Previously the business part was a bit concealed during our meetings and we were talking about it unofficially and today we have a meeting dedicated to our core business" (Bus AM#3)

"Sometimes during the meetings we were talking about the business part which didn't interest us particularly. Today our meetings are based on retail market so we have only what we need to know" (Pers AM#2)

Thus, complementary to this building strategizing, managers had the opportunity to innovate regarding the animation and the content transmitted during these meetings. One manager asked some external experts to intervene on specific topics, while the other invited employees to share their expertise and explained how they managed to reach their objectives. For example, during an observation of a retail market sales meeting, one employee took the floor and described the consequences of the 2018 new financial law for the customers and provided advice on what they could offer to different categories of customers.

These dwelling micro-strategizing progressively changed the dynamics of the discussions during the sale meetings:

"on a file presented by my fellow, my manager had a reserved opinion […] but he did not give his opinion, he kept it to himself and asked us to give our opinion on that file, we exchanged all three of us, then he gave his opinion and we concluded together" (Bus AM#5).

"It is important for the business market, even if he will never be in charge on this client, it is important because the manager side for the client is a real plus. He is telling himself 'Ok I know the manager', it valorizes them. This is why he [BM] will meet all my big customers" (Bus AM#2)

Overall, the data suggested that co-animation was a constructive strategy that nurtured account managers' skill levels through empirically based personal feedback. Co-animation also provided the opportunity to discuss technical issues and to provide a better service and improved customer satisfaction.

Adjust managers activity according to employees' needs

The third main objective driving the project was to free managers from time-consuming administrative tasks to enable them to focus on their managerial duties.

To do so, the prescribed distinction between retail and business market was respected, as was the risk delegation within the management team to reduce their workload.

"before [our BM] was seeing everyone in the office so he was not always available, today with

[our ABM] it is easier because obviously she is more focus on us, she has only us to care about whereas we had to share [our BM] with everybody" (Pers AM#4) "the employee may not come to see you because you are overwhelmed by others things and you may not be available for him, with the new organization we won't be in the same configuration.

Tomorrow we will be much more likely to help him and give him a more precise solution. So it's win-win" (BM#1)

This fostered managers visibility and mobility and allow them to adjust their activity according to the different needs of the local context:

"at the beginning of every week [our BM and ABM] send us a planning for one or two weeks of 'who is where ' and 'who does what', it's good because at least we know when there are meetings or absences" (Pers AM#6)

This latitude encourages managers' dwelling micro-strategizing. We observed that managers' mobility was initially organized by half-days to ensure they visited a banking branch several times each week but then the employees mentioned that it could be more appropriate for managers to stay in one place during an entire day in order to allow them to more easily have time to see a manager:

"It's more flexible when he's here during the whole day because there is always a moment of relief whereas when he [BM] stays for only 3 hours, I can have an appointment or my colleague can go and see him and then he has less time for me" (Bus AM#2) "When he is here for the day, you can overflow a little bit on the midday break but when he must go somewhere else, he [BM] can't take 30 minutes during his break because he has the journey and even if we have phones or e-mails, there are cases that we need to talk in person" (Bus AM#3)

The consequence was that managers could also better adapt their activity depending to the needs of the particular banking branch. For example, one banking branch was without a receptionist, so the account manager had to welcome the customers during his appointment. After the reengineering of the bank's structure, the management team decided to visit three days each week to ensure this reception role was covered and the account manager became more available for his scheduled appointments and sales activities. The following interview excerpt explains:

"Every Wednesday we have the operational manager coming to the agency, every Thursday we have the assistant director coming and every Saturday morning the branch manager coming, so I would say that we are very few times alone.

[…] And when we have an appointment and there is a client who shows up at the reception desk, they take care of them" (Pers AM#8).

Concerning the individual interviews, empowering middle manager enable them to globally respect the weekly frequency:

"I have a global vision but the time I have [in the old organization] does not let me be sufficiently present behind each advisor. Tomorrow, as part of the business project […] the weekly interviews,

which are not always weekly, or not done, or not always done well, because of lack of time in the daily organization, will become regular so there will be better advisor steering." (BM#1)

"From time to time we told them [our team]: we will assist and support you, but we never did it because we didn't have time; now we will." (BM#2)

"Before [in the old organization], I used to do episodic individual support. Now we can keep a calendar of the individual meetings and be more frequently present" (BM#2)

However, middle managers' dwelling strategizing led them to adjust the priorities of these appointments according to their employees' needs. Many of them took advantage of the opportunity to discuss complex files rather than focusing on their professional practices, as recommended by the new organization. As we see in the excerpts below, some account managers reported retaining non-urgent files to discuss during these interviews with their manager. This was an example of how one practice in the recommended organizational model evolved, widening in focus from managerial support to include discussions designed to enhance the customer relationship -the wider focus of the account managers' professional activity.

"During this weekly interview, we can submit a case, if we have questions this is the moment, then

we also debrief about the activity of the week" (Pers AM#2)

"My interview occurs every week on Thursday morning so we fixed a moment and we take this opportunity to speak about specific cases and validate them together. It's an occasion to discuss, so after the cases, we provide an overview on my results and objectives" (Bus AM#6) "I start each individual interview like that, no matter who the employee is, it is first of all his expectations, his grievances, what annoyed him for the last 15 days. It can be about everything and then it actually takes 10-15 minutes to process and they can arrive with a presentation files on which we are going to exchange" (BM#1).

The analysis of managers' change translating micro-practices were all designed to achieve the three main objectives of the organizational project but they reveal a clear combination between dwelling and building strategizing (see Figure 2 above). This can be explained by the fact that key principles of the organizational change project had already been assimilated by all managers as a result of a continuous communication campaign, both oral and material, linked to the project.

DISCUSSION AND CONCLUSION

When facing increasing competition, organizations tend to refine their structures to gain agility and competitive edge, calling into question the legitimacy of middle managers [START_REF] Carney | Freedom, Inc: How Corporate Liberation Unleashes Employee Potential and Business Performance[END_REF][START_REF] Hamel | First, let's fire all the managers[END_REF][START_REF] Lee | Self-managing organizations: exploring the limits of lesshierarchical organizing[END_REF]. Other scholars have questioned the prudence of such approaches underlining the strategic role of middle managers (Arnaud, Mills, & Legrand, 2016;[START_REF] Detchessahar | Santé au travail. Quand le management n'est pas le problème, mais la solution[END_REF][START_REF] Rouleau | Micro-Practices of Strategic Sensemaking and Sensegiving: How Middle Managers Interpret and Sell Change Every Day[END_REF]. In this article, we have examined what happens when an organization not only endorses the need for middle managers but also gives them greater latitude to engage in strategic organizational change. We see our contributions as threefold.

First, this study contributes to the rapidly developing and influential scholarship on strategy as practice (SaP) [START_REF] Burgelman | My Once and Future Partner-or Acquiree A Mechanisms-Based, Policy-Capturing Analysis[END_REF][START_REF] Golsorkhi | Cambridge handbook of strategy as practice[END_REF][START_REF] Rouleau | What is strategy-as-practice[END_REF][START_REF] Whittington | The work of strategizing and organizing: for a practice perspective[END_REF][START_REF] Whittington | Strategy as practice, process, and institution: Turning towards activity[END_REF], providing further evidence that strategizing is an ongoing flow of practices constructed by practitioners through their praxis in situ but aligned with strategic decisions taken by top management. Specifically, our empirical case study demonstrates how middle managers were instrumental in instigating strategic practices prescribed by top management's as well as contributing to spontaneous and undeliberated adaptations of the strategic prescriptions in order to achieve a better fit between frontline practice and the local context. These two types of strategy in practice mirror [START_REF] Chia | Strategy as practical coping: a Heideggerian perspective[END_REF] distinction between building and dwelling visions of strategizing. Our results show that building strategizing preceded dwelling strategizing in order to allow managers to apply without delay top-managers' prescriptions in the first stage of change implementation but did not preclude transformation of these later if they were found not to fit with local circumstances. Moreover, some of these emergent strategic practices then became the new 'rules' as they were perceived as particularly pertinent by top-management. This result contrast with the assumptions of the strategy emergence sequence (Chia & Holt, 2006, p. 641;Chia & MacKay, 2007, p. 230), by showing that it is also conceivable to think of a retroactive relationship between building and dwelling strategizing (see Figure 3 below). This possibility was demonstrated empirically in our case by the way the prescriptions from the top management were implemented, as a form of planned organizational change [START_REF] Rosenbaum | Planned organisational change management: Forward to the past? An exploratory literature review[END_REF], and then adapted and transformed repeatedly to accommodate the emergent subjectivities of local practice. This iterative nature of strategic practice allowed the final outcome to be a highly refined blend of prescribed intervention and adaptation driven simultaneously by prospective and retrospective sensemaking as shown in the figure below (Figure 3).

Figure 3. Prescribed and adaptive strategizing in change translation

Secondly, in opposition with mainstream managerial innovation discourses which propose selfmanagement as a substitution for middle management [START_REF] Carney | Freedom, Inc: How Corporate Liberation Unleashes Employee Potential and Business Performance[END_REF][START_REF] Hamel | First, let's fire all the managers[END_REF][START_REF] Hamel | The future of Management[END_REF][START_REF] Magpili | Self-Managing Team Performance: A Systematic Review of Multilevel Input Factors[END_REF], our study emphasizes middle manager's strategic role during organizational change (Arnaud, Mills, & Legrand, 2016;[START_REF] Radaelli | Middle managers and the translation of new ideas in organizations: a review of micro-practices and contingencies[END_REF][START_REF] Rouleau | Middle managers, strategic sensemaking, and discursive competence[END_REF][START_REF] Teulier | Middle managers' sensemaking and interorganizational change initiation: translation spaces and editing practices[END_REF]. Our article thus adds an original case study to the scant research addressing the strategic value of middle managers emphasizing the idea that managers are not only change passive 'translators' but that they enact a real agency in the organizational change process.

Finally, from a managerial perspective, our findings enable to enlight what empowering middle managers means [START_REF] Buick | Enabling Middle Managers as Change Agents: Why Organisational Support Needs to Change[END_REF][START_REF] Gomez | The leader-member exchange as a link between managerial trust and employee empowerment[END_REF]. Indeed, we show clear empirical illustrations of how middle managers can be empowered by both organizational structure and topmanagement support. Our results also allow to better understand how middle managers can foster their teams' development if empowered by top management to embrace their strategic role. Our findings indeed provide three types of strategic activities meant to encourage employees' empowerment: (1) federate the team spirit to facilitate collaboration; (2) develop employees' capabilities, and (3) adjust managers' activity according to employees' needs. These results go beyond the banking sector and allow to give an empirical illustration of the progressive development of empowering leadership micro-practices [START_REF] Cheong | Two faces of empowering leadership: Enabling and burdening[END_REF][START_REF] Li | Linking empowering leadership and change-oriented organizational citizenship behavior: The role of thriving at work and autonomy[END_REF][START_REF] Türkmendağ | Empowering leadership and knowledge management: the mediating role of followers' technology use[END_REF].

Limitations and recommendations for future research

Our research was an in-depth case study and as such its findings cannot be generalized. Instead, its value lies in how the empirical analysis it offers provides a basis for further study. Firstly, it encourages to pursue our research on the meager literature on middle manager empowerment [START_REF] Block | The empowered manager: Positive political skills at work[END_REF][START_REF] Quinn | The road to empowerment: Seven questions every leader should consider[END_REF], by providing an original and finely nuanced case that reveals what this entails in practice in a large organization. Secondly, studying the implementation of a managerial team, our case study highlights the need to investigate further the practices that encourage leadership to be enacted as a distributed performance, shared across a management team [START_REF] Brown | Antecedents of shared leadership: empowering leadership and interdependence[END_REF][START_REF] Pearce | Shared leadership: reframing the hows and whys of leadership[END_REF]. These practices are more and more diffused, and their consequences remain under-explored [START_REF] Sweeney | Shared leadership in commercial organizations: A systematic review of definitions, theoretical frameworks and organizational outcomes[END_REF][START_REF] Zhu | Shared leadership: A state-of-the-art review and future research agenda[END_REF]. Following this perspective, future research could be interested in the evolution of the identity perceived by managers of their own role when it is impacted by organizational changes [START_REF] Crook | Leader,"know yourself": bringing back selfawareness, trust and feedback with a theory O perspective[END_REF], especially in times of increasing remote work or the introduction of algorithmic management [START_REF] Galière | When food-delivery platform workers consent to algorithmic management: a Foucauldian perspective[END_REF]

  depicts the previous structure on the left and this new structure on the right.

Figure 1 .

 1 Figure 1. The organization structure before and after the organizational re-engineering

  The interviewees were chosen to represent each of the banking branch functions and were contacted during the observations in order to obtain a heterogeneous sample in terms of gender, age and professional tenure (see Appendix 1). In total, we conducted 32 interviews in French in the employees' personal offices with an average length of 43 minutes (see Appendix 1), producing 310 pages of transcription. The verbatims used in the findings' session were then translated English by one of the co-authors and checked by the other co-author.The data analysis was operated in two stages. First, one of the co-authors conducted an open coding of the observations field notes and interview transcripts to list and categorize all the middle managers practices aimed at translating the organizational change. This first open-coding process has then been discussed by the two other co-authors and ensured data saturation as the last interviews analyzed did not provide any new codes. By doing so, twelve translating micro-practices were identified, grouped in three main categories emerged from the results, suggesting the consequences of these practices: (1) federate the team spirit to facilitate collaboration across the branches of a unit; (2) develop employees' capabilities to improve customer satisfaction, and (3) adjust managers' activity according to employees' needs so employee's role engagement, wellbeing and productivity were optimized (see Figure2below on the left). These three types of translating micro-practices were the first step of our findings, enabling us to answer our research question(Merriam & Tisdell, 2015, p.203).

Figure 2 .

 2 Figure 2. Empowered middle managers' micro-strategizing during organizational changeWe then focused on how organizational change translation practices were enacted, distinguishing

Data collection and analysis

The qualitative data were collected from three different sources so that we gain a finely nuanced appreciation of not only actions and outcomes, but the circumstances in which these emerged.

Prior to the project implementation, we firslty analyzed a set of internal documents such as the organizational chart of the banking branches and the job descriptions for each functions, as well as the organizational change booklet distributed to all of the company's members which introduces the motivations, the main objectives and the implementation timeline of the organizational project.

The analysis of these secondary data enabled us to examine the top-management discourse explaining how the proposed organizational change was designed to address the strategic issues the company was facing, which justified the strategic nature of the transformations operated. It also allowed us to identify precise language elements that facilitate our interaction with the work teams, and thus ease our integration in the field. We then conducted 4 exploratory interviews with one top manager, the leader of the project and two region managers (see Appendix 1). These interviews were open ended aiming at understanding the organizational change context and gathering managers' prospective sense of the proposed organizational change. After the start of the organizational project implementation, we conducted 11 hours of non-participatory observations in the two different banking branches in order to capture in situ middle managers' change translation practices (Merriam & Tisdell, 2015, p. 139). These observations were recorded as field notes [START_REF] Van Maanen | Tales of the field: On writing ethnography[END_REF], to better understand the "situated activity" of strategy practitioners [START_REF] Johnson | Micro strategy and strategizing : towards an activity-based view[END_REF][START_REF] Whittington | The work of strategizing and organizing: for a practice perspective[END_REF]. These observations also led to informal discussions and helped to gain employees' trust who feared that the presence of researchers would be used to ensure the proper implementation of the top management prescriptions. The PhD status of one of the coauthors on the field also helped to reassure interviewees that the study was undertaken

"we validate technically between us our mortgage files so it allows us to exchange on our practices so it's still rather interesting to do this way even if everyone has their own work as well" (Pers AM#2).

As the above testimonies illustrate, this strategic practice allows both to discuss precise customers' issues and to enhance team collaboration by sharing practices.

Finally, the co-animation between an account manager and the (assistant) branch manager during customer appointments was progressively implemented as planned by top-management. This building strategizing allowed the account manager to receive feedback on their performance while the branch managers gained first-hand experience of the customer interface. The following interview excerpts describe how this co-animation was experienced as a source of useful performance feedback:

"It [customer appointment with the manager] is like the camera when you interpret a role play and then you watch your performance. You have someone next to you, like a mirror that provide his expertise 'you should avoid this king of things, you should go this way before going straight to the solution'" (BM#1) "Since we debrief on the interviews, it gives us his opinion on the way we get into some issues and his feeling about what can be improved" (Bus AM#8) This strategic practice was also applied to satisfy or legitimize the client rather than to shadow employees as they performed their tasks:

"He [the ABM] offered to do some with but they were important client with specific issues so he has been involved in the appointment. […] No it was not for me, he was just here to support the proposition we made" (Pers AM#7)

DISCLOSURE OF INTEREST

The authors report no conflict of interest.

APPENDIX A. INTERVIEWS INFORMATION