Charlotte Dubois

Artem Kondratskyi

Gabriel Bidaux

Lucile Noyer

Eric Vancauwenberghe

Valério Farfariello

Robert-Allain Toillon

Morad Roudbaraki

Dominique Tierny

Jean-Louis Bonnal

Natalia Prevarskaya

Vanden Fabien

Abeele

Fabien Vanden

Affiliations

Journal Pre-proof CO-TARGETING MITOCHONDRIAL CA 2+ HOMEOSTASIS AND AUTOPHAGY ENHANCES CANCER CELLS CHEMOSENSITIVITY

Keywords: Calcium, apoptosis, mitochondria, autophagy, chemoresistance, ex-vivo tumor models, xenograft mouse models, combined therapy

Mitochondria are important cell-death checkpoints and mitochondrial Ca 2+ overload is considered as a potent apoptotic intrinsic pathway inducer. Here, we report that this Ca 2+ apoptosis link is largely ineffective in inducing cell-death just by itself and required a concomitant inhibition of autophagy to counteract its pro-survival action. In such condition, an acute mitochondrial stress revealed by a DRP1 mediated mitochondrial dynamic remodeling is observed concomitantly with mitochondrial depolarization, release of cytochrome c and efficient apoptosis induction. We also uncover that mitochondrial Ca 2+ status modulates the function of autophagy as a sensitizer for chemotherapies. This priming mediated by mitochondrial Ca 2+ overload and inhibition of autophagy sensitizes many cancer cells types to different chemotherapies with independent mechanisms of action. Collectively, our results redefine an important cell signaling pathway, uncovering new combined therapies for the treatment of diseases associated with mitochondrial Ca 2+ homeostasis disorders such as cancer.

Introduction

Calcium (Ca 2+ ) is a critical intracellular second messenger, which controls key cell fate decisions, such as metabolism, proliferation or apoptosis [START_REF] Rossi | Calcium, mitochondria and cell metabolism: A functional triangle in bioenergetics[END_REF]. Specific Ca 2+ signaling pathways have long been known to play an important role in apoptosis induction and/or regulation, which occurs through endoplasmic reticulum (ER) Ca 2+ stress, cytosolic and mitochondrial Ca 2+ overload [START_REF] Berridge | The versatility and universality of calcium signalling[END_REF][START_REF] Orrenius | Regulation of cell death: the calciumapoptosis link[END_REF][START_REF] Pinton | Calcium and apoptosis: ER-mitochondria Ca2+ transfer in the control of apoptosis[END_REF]. Each of these pathways has been associated alone or in combination with apoptosis induction or resistance, both in physiological and physiopathological conditions. Indeed, the ER lumen is the major storage of intracellular Ca 2+ , allowing the proper folding of proteins by Ca 2+binding chaperones [START_REF] Wang | The impact of the endoplasmic reticulum protein-folding environment on cancer development[END_REF]. Depletion of ER Ca 2+ stores is considered to be a robust stress condition that triggers cell death [START_REF] Giorgi | PML regulates apoptosis at endoplasmic reticulum by modulating calcium release[END_REF][START_REF] Prevarskaya | Ca2+ homeostasis in apoptotic resistance of prostate cancer cells[END_REF]. Other studies have also demonstrated that a large and sustained elevation of cytosolic Ca 2+ concentration ([Ca 2+ ] c ) is another key process for triggering apoptosis, mainly induced by store-operated Ca 2+ entry (SOCE) [START_REF] Vanden Abeele | Bcl-2-dependent modulation of Ca(2+) homeostasis and store-operated channels in prostate cancer cells[END_REF][START_REF] Prevarskaya | Ion channels and the hallmarks of cancer[END_REF].

Finally, mitochondrial Ca 2+ overload resulting from sustained elevation of [Ca 2+ ] c is considered to be one of the ways to induce mitochondrial-mediated intrinsic apoptotic pathway [START_REF] Berridge | The versatility and universality of calcium signalling[END_REF][START_REF] Orrenius | Regulation of cell death: the calciumapoptosis link[END_REF][START_REF] Pinton | Calcium and apoptosis: ER-mitochondria Ca2+ transfer in the control of apoptosis[END_REF][START_REF] Zhivotovsky | Calcium and cell death mechanisms: a perspective from the cell death community[END_REF]. The crucial factor is the magnitude of the Ca 2+ signal received by the mitochondria. A high Ca 2+ load will lead to the mitochondrial permeability transition pore (MPTP) [START_REF] Lemasters | Mitochondrial calcium and the permeability transition in cell death[END_REF] and eventually to the permeabilization of the outer mitochondrial membrane (OMM). As a consequence, targeting mitochondria and in particular mitochondrial Ca 2+ homeostasis to sensitize cancer cells to apoptosis and to overcome drug resistance, represents a major aim in anti-cancer therapy [START_REF] Wen | Targeting cancer cell mitochondria as a therapeutic approach[END_REF][START_REF] Zhang | Targeting mitochondrial biogenesis to overcome drug resistance to MAPK inhibitors[END_REF].

However, the expected role of Ca 2+ could be less clear than it has first been proposed. Indeed, we have identified in a previous study, that a potent Ca 2+ homeostasis disruption characterized by a sustained increase of cytosolic Ca 2+ and endoplasmic-reticulum (ER) Ca 2+ stress is not sufficient enough to induce apoptosis [START_REF] Dubois | Differential effects of thapsigargin analogues on apoptosis of prostate cancer cells: complex regulation by intracellular calcium[END_REF]. However, the role of mitochondria was not investigated and more importantly the molecular mechanisms underlying this atypical mode to escape from cell-death were not identified. Moreover, an increasing number of studies have demonstrated that Ca 2+ also plays an important role in the regulation of more recent hallmark of cancer, such as autophagy involved in tumor development and cancer therapy [START_REF] Degenhardt | Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis[END_REF][START_REF] Hanahan | Hallmarks of Cancer: The Next Generation[END_REF][START_REF] Yang | The Role of Autophagy in Cancer: Therapeutic Implications[END_REF].

Autophagy is a catabolic degradation process in which cellular proteins and organelles are enveloped by double-membrane autophagosomes and degraded in lysosomes [START_REF] Chen | The regulation of autophagy -unanswered questions[END_REF][START_REF] Ravikumar | Regulation of mammalian autophagy in physiology and pathophysiology[END_REF]. The autophagic process attempts to restore metabolic homeostasis and the current consensus is that autophagy has a dual role in cancer [START_REF] Jiang | Autophagy in cellular metabolism and cancer[END_REF][START_REF] Kroemer | Autophagy: a druggable process that is deregulated in aging and human disease[END_REF][START_REF] White | The role for autophagy in cancer[END_REF]. During the early steps of cancer development, autophagy functions as a tumor suppressor mechanism by preventing the accumulation of damaged organelles and aggregated proteins. In contrast, autophagy is considered as a prosurvival mechanism for established tumors in response to metabolic stresses such as nutrient deprivation, hypoxia, and absence of growth factors. Autophagy has also emerged as a crucial player in drug resistance in response to chemotherapies [START_REF] Kroemer | Autophagy: a druggable process that is deregulated in aging and human disease[END_REF]. Importantly, autophagy is shown to precede or to act in parallel with apoptosis process but the precise molecular mechanisms involved are still not fully understood [START_REF] Sui | Autophagy and chemotherapy resistance: a promising therapeutic target for cancer treatment[END_REF] and in particular the role of autophagy in cell-death mediated by Ca 2+ homeostasis disruption is not fully understood. Finally, it has been also reported that some Ca 2+ signaling pathways and Ca 2+ modulators used to depict apoptotic pathways have also significant implications in other signaling pathways such as autophagy, highlighting the need of better tools [START_REF] Bootman | The regulation of autophagy by calcium signals: Do we have a consensus?[END_REF][START_REF] Dubois | The calcium-signaling toolkit: Up-dates needed[END_REF].

Here, we dissect the interplay between survival pathways such as autophagy and mitochondrial dynamics to establish their precise role(s) in cell death mediated by Ca 2+ homeostasis disruption. Collectively, our results challenge a crucial paradigm in cell death and provide new concepts for more rational approaches in cancer treatments. Indeed, we propose an effective combined strategy based on concomitant mitochondrial Ca 2+ overload and autophagy inhibition with drugs in clinical evaluation to increase chemotherapies sensitivity in most common cancer types.

Results

Ca 2+ homeostasis perturbations per se are not sufficient to induce apoptotic cell death

To investigate the role of Ca 2+ homeostasis perturbations in apoptosis induction, we performed an in-depth study by using several sarcoplasmic endoplasmic reticulum Ca 2+ ATPase (SERCA) inhibitors (Thapsigargin (TG), TG analogues and cyclopiazonic acid (CPA)). SERCA resides in the endoplasmic reticulum (ER) and plays a crucial role in maintaining cellular Ca 2+ homeostasis [START_REF] Berridge | The versatility and universality of calcium signalling[END_REF][START_REF] Orrenius | Regulation of cell death: the calciumapoptosis link[END_REF][START_REF] Pinton | Calcium and apoptosis: ER-mitochondria Ca2+ transfer in the control of apoptosis[END_REF]. We used concentrations of SERCA inhibitors known to induce a robust disruption of Ca 2+ homeostasis [START_REF] Quynh Doan | Thapsigargin, Origin, Chemistry, Structure-Activity Relationships and Prodrug Development[END_REF]. The TG analogues tested were ASP-8ADT (also known as 12-ADT-ASP), EPO-8ADT and LEU-8ADT, and all of them have been characterized in cell free system for their specific activity on SERCA pumps and functionally on several cell types [START_REF] Jakobsen | Design, synthesis, and pharmacological evaluation of thapsigargin analogues for targeting apoptosis to prostatic cancer cells[END_REF]. As we have already demonstrated [START_REF] Dubois | Differential effects of thapsigargin analogues on apoptosis of prostate cancer cells: complex regulation by intracellular calcium[END_REF], all analogues were able to induce an influx of Ca 2+ across the plasma membrane due to the activation of SOC (Store Operated Channel) in response to ER Ca 2+ store depletion triggered by SERCA inhibition (Figure 1A). Here, we therefore evaluated their ability to maintain high cytosolic Ca 2+ concentration ([Ca 2+ ] c ) over a longer period of time which has been also associated with apoptosis induction (Figure 1B) [START_REF] Berridge | Calcium signalling: dynamics, homeostasis and remodelling[END_REF][START_REF] Clapham | Calcium Signaling[END_REF]. We observed that only TG, ASP-8ADT and LEU-8ADT (1 µM/ 18 hr) were able to induce a sustained [Ca 2+ ] c increase compared to EPO-8ADT or CPA (1 µM/ 18 hr) (Figure 1B). In accordance, these discrepancies may have significant consequences on mitochondria Ca 2+ contents, as it is largely accepted that sustained Ca 2+ entry is also a major factor in the process of mitochondrial Ca 2+ overload [START_REF] Berridge | The versatility and universality of calcium signalling[END_REF][START_REF] Orrenius | Regulation of cell death: the calciumapoptosis link[END_REF][START_REF] Pinton | Calcium and apoptosis: ER-mitochondria Ca2+ transfer in the control of apoptosis[END_REF][START_REF] Zhivotovsky | Calcium and cell death mechanisms: a perspective from the cell death community[END_REF]. Thus, we evaluated the extent of mitochondrial Ca 2+ overload following the same setting of treatment. We observed that, only TG, ASP-8ADT and LEU-8ADT induced mitochondrial Ca 2+ overload in prostate cancer cell lines (Figures 1C,D) and 5 additional cancer cell lines (Supplementary Figure 1). In parallel, we correlated the ability of these compounds to induce Ca 2+ mediated apoptosis in prostate (Figures 1E,F,G,H), breast (Figure 1I) and pancreatic cancer cells (Figure 1J) (Supplementary Figure 1A-O). Surprisingly, only TG and LEU-8ADT were potent apoptosis inducers in all cell lines. Both compounds have a similar ability to induce mitochondrial Ca 2+ overload and to maintain high [Ca 2+ ] c over a longer period (Figure 1B). However, LEU-8ADT induced a slower initial Ca 2+ influx rate compared to TG (Figure 1A). Conversely, the initial Ca 2+ influx rate was higher for ASP-8ADT compared to LEU-8ADT despite its low efficiency in inducing cell death. This implies that the initial Ca 2+ influx was not correlated with the induction of apoptosis. Concerning TG and ASP-8ADT, while the remodeling on Ca 2+ homeostasis was quite similar, compared to TG, apoptosis induced by ASP-8ADT was very modest and particularly in the LNCaP or PC-3 prostate cancer cell lines. These results were unexpected as they are not in line with the commonly established key role of mitochondrial Ca 2+ overload in the process of apoptosis induction. Thus, we decided to confirm that ASP-8ADT was able to induce mitochondrial Ca 2+ overload by using two other independent approaches that allow a direct assessment of Ca 2+ handling by mitochondria. By measuring steady-state Ca 2+ concentration in mitochondria with the 4mtD3cpv biosensor or with the ratiometric genetically encoded Ca 2+ probe mito-GEM-GECO1, we confirmed that TG and ASP-8ADT induced similar mitochondrial Ca 2+ overload (Figure 1K) (Supplementary Figures 1P-R and methods section).

Importantly, at the micro-molar concentration (1 µM) used in the present study, both TG and ASP-8ADT totally inhibit SERCA activity [START_REF] Winther | Critical Roles of Hydrophobicity and Orientation of Side Chains for Inactivation of Sarcoplasmic Reticulum Ca2+-ATPase with Thapsigargin and Thapsigargin Analogs[END_REF]. Thus, differences observed in apoptosis induction were not correlated to differential action or affinity on SERCA activity.

Moreover, in a previous study [START_REF] Dubois | Differential effects of thapsigargin analogues on apoptosis of prostate cancer cells: complex regulation by intracellular calcium[END_REF], we already observed that ASP-8ADT did not cause as much cell death during chronic exposure and we attributed this effect to a weaker ability of the ASP-8ADT compound to keep the ER stores depleted. However, this conclusion is ruled out in the present study, as we were unable to detect differences in the hallmarks of Ca 2+ changes induced by TG or ASP-8ADT over a longer period covering the whole duration of apoptosis induction phase. We also checked that the uncoupling between Ca 2+ homeostasis disruption and apoptosis induction did not result from impairments in ER stress response (Supplementary Figures 1S-Y) or Reactive Oxygen Species (ROS) accumulation in response to mitochondrial Ca 2+ overload mediated by ASP-8ADT (Figure 1L). This in-depth study suggests the involvement of an undefined step/factor needed for an efficient cell death induction in response to Ca 2+ homeostasis disruption and following mitochondrial Ca 2+ overload.

Mitochondrial Ca 2+ overload induces a partial MPTP opening, ineffective to induce mitochondrial membrane depolarization and cyt c release

It is generally considered that mitochondrial Ca 2+ overload induces continuous MPTP openings during cell death leading to loss of both Ca 2+ and proton gradients across the inner mitochondrial membrane [START_REF] Baumgartner | Calcium elevation in mitochondria is the main Ca2+ requirement for mitochondrial permeability transition pore (mPTP) opening[END_REF][START_REF] Giorgi | Mitochondrial Ca(2+) and apoptosis[END_REF]. We used the gold standard calcein/Co 2+ -quenching technique, which allows a quantification of MPTP opening [START_REF] Bonora | Comprehensive analysis of mitochondrial permeability transition pore activity in living cells using fluorescence-imaging-based techniques[END_REF]. We showed that a 18 hr pretreatment with either TG (known to promote MPTP opening [START_REF] Korge | Thapsigargin directly induces the mitochondrial permeability transition[END_REF]) or ASP-8ADT, were associated with a similar cobalt-mediated quenching of calcein fluorescence (Figures 2A,B) suggesting that MPTP was opened to an equivalent extent. This MPTP opening was abolished by a 1hr-pretreament with cyclosporine A (CsA; 0,5 µM), an inhibitor of MPTP opening which binds cyclophilin D.

We also tested ionomycin (IM; 1 µM), a Ca 2+ ionophore known to trigger mitochondrial Ca 2+ overload and activation of MPTP in different cell models [START_REF] Chaudhuri | Mitochondrial calcium uniporter regulator 1 (MCUR1) regulates the calcium threshold for the mitochondrial permeability transition[END_REF][START_REF] Ying | Short-Term Mitochondrial Permeability Transition Pore Opening Modulates Histone Lysine Methylation at the Early Phase of Somatic Cell Reprogramming[END_REF]. We observed with IM a potent activation of MPTP compared to TG and ASP-8ADT (Figure 2C). In accordance, CsA pre-treatment (1 hr before TG or ASP-8ADT treatments; 0,5 µM) induced an increase of mitochondrial Ca 2+ content in response to TG or ASP-8ADT (Figure 2D). We concluded from these experiments that both ASP-8ADT and TG were able to induce similar but not maximal MPTP opening (as observed with IM). These experiments challenge the role of MPTP openings and mitochondrial Ca 2+ overload implying that another process impaired cell death induction with ASP-8ADT. As a readout of cell death and MPTP openings, we evaluated the release of cyt c (Figures 2E,F) (Figures S2A,B) and the loss of mitochondrial membrane potential (Figures 2G,H). Indeed, cyt c release from mitochondria and loss of mitochondrial membrane potential (∆Ѱm) are considered to occur subsequently to continuous pore activation. Our results clearly showed that ASP-8ADT failed to induce these features compared to TG and this, despite its ability to induce both similar mitochondrial Ca 2+ overload and MPTP opening. We also clearly demonstrated that TG induced only a partial mitochondrial depolarization in comparison to the mitochondria uncoupler FCCP (1 µM) (Figures S2C,D). Our results also demonstrated that IM is a more potent inducer of MPTP opening compared to either ASP-8ADT or TG (Figure 2C). Thus, this partial MPTP opening induced by ASP-8ADT may explain that mitochondrial membrane potential is preserved with ASP-8ADT. Indeed, MPTP opening is intrinsically depolarizing, but we clearly showed that only partial MPTP opening occurs. Moreover, a situation with no mitochondrial Ca 2+ overload occurred only when mitochondrial potential is totally collapsed by using FCCP as pretreatment (Figure 2D). Taken together, these results suggest that partial MPTP opening resulting from mitochondrial Ca 2+ overload and ROS accumulation is not enough to induce loss of mitochondrial membrane potential and release of cyt c.

Autophagy inhibition is a pre-requisite for mitochondrial depolarization leading to apoptosis mediated by Ca 2+ homeostasis disruption TG-treated cells exhibited mitochondrial membrane depolarization and efficient apoptosis induction, suggesting a direct link with its apoptotic potency. Interestingly, it has been reported that TG has significant implications in other Ca 2+ -mediated and Ca 2+ -independent signaling pathways such as autophagy [START_REF] Bootman | The regulation of autophagy by calcium signals: Do we have a consensus?[END_REF][START_REF] Decuypere | A dual role for Ca2+ in autophagy regulation. Cell Calcium, Special issue on Ca2+ signaling mechanisms of cell[END_REF]. We hypothesized that apoptosis mediated by TG could be related to the inhibition of survival pathways such as autophagy. We used, an established strategy, a tandem-tagged GFP-mCherry-LC3 probe that can determine whether an autophagosome has fused with a lysosome, based on the distinct chemical properties of GFP and mCherry fluorophores [START_REF] Kimura | Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3[END_REF]. Under non-lysosomal and near-neutral pH conditions, both GFP and mCherry fluoresce. However, the low pH in the lumen of the lysosomes quenches the GFP signal, but not the mCherry. Using this approach, we found that upon TG treatment (2 hr, 1 µM) in LNCaP cells, multiple autophagosomes are visible without GFP quenching, thereby suggesting that fusion with lysosomes did not occur (Figures 3A,B). In contrast, upon ASP-8ADT treatment (2 hr, 1 µM), only mCherry-positive structures have accumulated. The addition of chloroquine (CQ) (prevents endosomal acidification leading to an inhibition of lysosome-endosome fusion) in association with ASP-8ADT mimics the autophagic signature observed with TG. Indeed, by quantifying the size of acidic vesicles formed after treatment with TG, ASP-8ADT alone or in combination with CQ, we observed an increase in size upon ASP-8ADT when combined with CQ to the same extent as TG (Figure 3C). In a second attempt, we evaluated the action of ASP-8ADT on the autophagic flux in LNCaP cells by using Western Blot analysis of LC3 I and II (formation of autophagosome) and P62 (autophagic flux) (Figures 3D,E,F). These results confirmed previous studies that reported a block of autophagy by TG, as revealed by both accumulation of P62 and LC3 II. These effects were drastically reduced with ASP-8ADT, thus revealing its inability to interfere with the autophagic process (Figures 3D,E,F) (see also Figure S3). We also confirmed the action of ASP-8ADT and TG on autophagic flux in PC-3 prostate cancer cells and we also evaluated their action on autophagic flux in conditions of autophagy induction induced by starvation (HBSS, 2 hr) (Figures S3A,B,C,D). These results confirmed that TG is able to block autophagy, as revealed by both accumulation of P62 and LC3 II. These effects were greatly reduced with the ASP-8ADT compound. We also confirmed by real-time PCR of P62 mRNA levels that P62 protein level in ASP-8ADT treated cells did not depend on variation of transcription (Figure S3D). We also checked the Beclin1 status (Figure S3E). We have further investigated the activity of TG on the autophagic process by using a more qualitative approach. By electron microscopy, we showed that only TG prevented the closure of the autophagosome and induced an accumulation of immature autophagosome that we never observed following ASP-8ADT treatment (Figure 3G).

We took advantage of the fact that ASP-8ADT did not exert an inhibitory effect on autophagy in LNCaP cells in order to study its implication on apoptosis induced by Ca 2+ homeostasis disruption. We used a combined treatment with an ER Ca 2+ stressor (TG or ASP-8ADT) and established autophagic inhibitors such as Chloroquine (CQ), Wortmanin (WT), Bafilomycin A1 (Baf A1) and a Rab7 inhibitory peptide. Hoechst staining experiments shown, using autophagy inhibitors, a 6-fold increase of ASP-8ADT potency to induce cell death (from 5% to 30%), without additional effect on TG potency after 48 hr of treatment (Figure 4A). By western blot we confirmed the cleavage of PARP (Figure S4F) as a marker of caspase-dependent apoptosis. We also performed immunostaining of cyt c on LNCaP cells treated with ASP-8ADT (1 µM, 18 hr) alone or in combination with the autophagic inhibitor Rab7 (100 nM).

Confocal images showed that ASP-8ADT induced the release of cyt c only upon autophagy inhibition (Figures S4A,B). We confirmed the requirement of autophagy inhibition with a genomic strategy by using siRNA against ATG5, which participates in the formation of au-tophagosome [START_REF] Ganley | Distinct autophagosomallysosomal fusion mechanism revealed by thapsigargin-induced autophagy arrest[END_REF]. Thereby, we showed that an early inhibition of the autophagic process by the down regulation of ATG5 leads to an increase in apoptosis induced by ASP-8ADT (Figure 4B). However, the percentage of apoptotic cells was not as high as with other inhibitors of autophagy (Figure 4A) or TG, because siRNA mediated knockdown of the autophagic regulatory gene ATG5 was only partial (Figure 4B,upper panel). By flow cytometry using the fluorescent mitochondrial membrane probe DiOC6(3), we demonstrated that Rab7 inhibitory peptide restored the ability of ASP-8ADT to induce mitochondrial membrane depolarization (Figure 4C). These results were confirmed using the JC-1 dye. We observed the progressive loss of red JC-1-aggregate fluorescence (polarized mitochondria) and a cytoplasmic diffusion of green monomer fluorescence (depolarized mitochondria) following exposure to ASP-8ADT in combination with Rab7 inhibitor (Figure 4D). We confirmed this specific point in prostate LNCaP, PC-3 and breast MCF-7 cancer cell lines (Figure 4D). Thus, these data clearly demonstrated that inhibition of autophagy is required for efficient mitochondrial membrane depolarization upon mitochondrial Ca 2+ overload leading to the release of cyt c and apoptosis induction. Finally, we investigated the potential role of BAX and BCL-2 proteins, two crucial actors involved in the mitochondrial apoptotic pathway, by studying their expression and subcellular distribution at mitochondria in LNCaP cells. Using subcellular fractionation technique, we observed that BAX level was nearly the same at mitochondria following treatments with TG or ASP-8ADT alone or in combination with autophagy inhibition. In contrast, BCL-2 expression was higher in all the conditions compared to control (Fig- ure 4E). These findings suggest that OMM permeabilization driven by BAX and BCL-2 interactions is not a critical event in apoptosis induced by TG or ASP-8ADT (in combination with autophagy inhibition). Thus, the weaker ability of ASP-8ADT to kill the cells could not be explained by the promotion of the BCL-2 survival pathway.

Concomitant inhibition of autophagy and mitochondrial Ca 2+ overload is required for inducing an acute mitochondrial stress revealed by a DRP1 mediated mitochondrial dynamic remodeling

We further explored the consequences of mitochondrial Ca 2+ overload and autophagy inhibition in the control of cell fate. It is well known that mitochondrial dynamics (fusion and fission) and autophagy, work together as a quality control mechanism in the life cycle of the mitochondrion [START_REF] Twig | Mitochondrial fusion, fission and autophagy as a quality control axis: the bioenergetic view[END_REF]. During the apoptotic process, mitochondrial networks are dramatically reorganized from long filamentous interconnected tubules into small punctate spheres. Whether remodeling of mitochondrial networks is necessary for apoptosis-associated cyt c release, or is merely an accompanying process, is still a subject of debate [START_REF] Sheridan | Mitochondrial fission/fusion dynamics and apoptosis[END_REF]. 3D reconstruction from confocal imaging experiments clearly showed that the filamentous interconnected tubules of the mitochondrial networks were preserved with ASP-8ADT alone but disrupted when combined with autophagy inhibitor as observed with TG (Figures 4F,G). These results clearly showed that mitochondrial Ca 2+ overload alone did not induce significant changes in mitochondrial dynamics suggesting that cell fate was not compromised when autophagy can play its pro-survival role. On the contrary, concomitant inhibition of autophagy forced the cell to use another pro-survival pathway mediated by mitochondrial dynamics. Indeed, recent studies have demonstrated that under sustained stress conditions, a DRP1-dependent mitochondrial fission is triggered and is involved in cellular survival at the early stage of injury [START_REF] Ikeda | New Insights into the Role of Mitochondrial Dynamics and Autophagy during Oxidative Stress and Aging in the Heart[END_REF][START_REF] Zuo | Mitochondria autophagy is induced after hypoxic/ischemic stress in a Drp1 dependent manner: the role of inhibition of Drp1 in ischemic brain damage[END_REF]. In this context, we showed that siRNA-mediated knockdown of DRP1 restored the apoptotic potential of ASP-8ADT (Figure 4H) confirming the crucial role of mitochondrial dynamics. siDRP1 treatments were functionally validated by confocal microscopy using MitoTracker Red CMXRos staining (via the observation of the characteristic mitochondrial hyperfusion) and western blot (Figure S4C,D,E). These results showed that a mitochondrial dynamic remodeling mediated by DRP1 is triggered and is involved in cell survival only when cells were exposed to concomitant Ca 2+ homeostasis disruption and autophagy inhibition. However, this prosurvival mechanism is not sufficient to prevent apoptosis induction induced by TG or ASP-8ADT in combination with autophagic inhibitors. These results also pointed out that autophagy and fission/fusion mitochondrial dynamics are two processes that could be used by the cell to counteract the mitochondrial stress mediated by Ca 2+ overload. These pro-survival pathways prevent mitochondrial collapse by inhibiting mitochondrial membrane depolarization. In fact, mitochondrial membrane depolarization seems to be the last and decisive event preceding apoptosis in response to mitochondrial Ca 2+ overload. We confirmed this feature by using FCCP (0,5 µM, 48 hr) to induce mitochondrial membrane depolarization and indeed, FCCP restored the ability of ASP-8ADT to induce cell death, whereas used alone it failed to do this significantly (Figures 4I,J).

Dual targeting of Ca 2+ homeostasis and autophagy improves chemotherapy efficiency in vitro and in vivo

Targeting mitochondria or autophagy to sensitize cancer cells to apoptosis is a major aim in anti-cancer therapy [START_REF] Jiang | Autophagy in cellular metabolism and cancer[END_REF][START_REF] Kroemer | Autophagy: a druggable process that is deregulated in aging and human disease[END_REF][START_REF] Wen | Targeting cancer cell mitochondria as a therapeutic approach[END_REF][START_REF] White | The role for autophagy in cancer[END_REF][START_REF] Zhang | Targeting mitochondrial biogenesis to overcome drug resistance to MAPK inhibitors[END_REF] and the medical and scientific community has also come to accept that monotherapy is unlikely to be the answer to completely destroy cancer cells [START_REF] Hanahan | Rethinking the war on cancer[END_REF]. Our results confirm that targeting mitochondrial Ca 2+ homeostasis alone displays only poor efficiency. The molecular basis underlying this lack of efficiency is that apoptosis mediated by mitochondria Ca 2+ overload requires inhibition of autophagy. This new interplay challenges a crucial paradigm in cell death and our results reveal novel combinatorial therapeutic strategies to improve clinical outcomes. ASP-8ADT is the active compound of the Mipsagargin family, a promising new anticancer treatment approved by the Food and Drug Administration (FDA) and used in several clinical trials (NCT01056029 and NCT01734681)) [START_REF] Denmeade | The SERCA pump as a therapeutic target: making a "smart bomb" for prostate cancer[END_REF][START_REF] Mahalingam | Mipsagargin, a novel thapsigargin-based PSMA-activated prodrug: results of a first-in-man phase I clinical trial in patients with refractory, advanced or metastatic solid tumours[END_REF]. Unfortunately, results are below expectations as published in a recent report [START_REF] Mahalingam | Mipsagargin, a novel thapsigargin-based PSMA-activated prodrug: results of a first-in-man phase I clinical trial in patients with refractory, advanced or metastatic solid tumours[END_REF]. Based on our fundamental findings we have evaluated using xenograft mouse models, cell line-based functional studies, and ex-vivo tumor models from clinical specimens, the relevance of our combined strategy to (1) increase the efficiency of ASP-8ADT in vivo (2) sensitize cancer cells to chemotherapies using a co-targeting strategy. Cancer-specific conventional chemotherapeutic agents have been used in combination with ER stressor agents and autophagy inhibition for six models of cancer: PC-3 (Figure 5A) and LNCaP (Figure 5B), primary human stromal prostate cancer cells (Figure 5F), MCF-7 and MDA-MB-231 breast cancer cells (Figures 5C,D) and MIA-PaCa2 pancreatic cancer cells (Figure 5E). Interestingly, in almost all conditions, the different autophagic inhibitors failed to significantly increase the chemotherapeutics apoptotic index. In contrast, ASP-8ADT used in combination with an autophagic inhibitor frequently increased chemotherapeutics cytotoxicity, regardless of their action mechanism, thus suggesting a unique way of priming cancer cells for several conventional cancer therapies. In the case of stromal prostate cancer cells and mammary cancer cells MCF-7, ASP-8ADT with autophagy inhibition induced high levels of apoptosis even in the absence of chemotherapy (Figures 5C,F). Most of our combined therapies were due to a synergic effect of the treatment (example of the analysis can be found in Figures 5G, H andS5A-C). Only few conditions were simply additive effects (pancreatic cell lines for cisplatin and 5-FU, cisplatin, docetaxel on MDA-MB-231).

We also conducted a series of combined therapy experiments on nude mice using prostate and breast tumor xenografts. The proof of principle concerning G-202 is well-established in vivo [START_REF] Denmeade | Engineering a prostate-specific membrane antigen-activated tumor endothelial cell prodrug for cancer therapy[END_REF] and in clinical trials, G-202 is injected intravenously. However, G-202 efficiency is also associated with an antiangiogenic effect that may biased ours results when used in in vivo experiments with chemotherapeutic agents such as taxotere (docetaxel). Thus, in our experiments, to achieve ER Ca 2+ stress, tumors were injected intratumorally (i.t.) on a basis of six injections over two weeks with ASP-8ADT alone (1 µM/PBS), or in combination with CQ (50 µM/PBS). The results of these experiments revealed that animals treated with a combined therapy displayed significantly less tumor growth than animals treated with ASP-8ADT alone (Figure 6A) associated with increased cell death, as revealed by TUNEL staining (Figure 6B) and PARP cleavage (Figure 6C). We also assessed the ability of the combined therapy in priming cancer cells to a classical chemotherapeutic agent such as docetaxel or 5-Fluoro-Uracile (5-FU) on respectively prostate and breast xenografts. To reduce the number of mice used, accordingly to our strict ethical rules, we have focused our experiment on proving the efficiency of the combined therapy and consequently did not assess docetaxel or 5-FU alone (based on the literature and strong in vitro data). The combined therapy (ASP-8ADT/CQ) again significantly increased the capacity of docetaxel and 5-FU to reduce tumor growth and increase apoptosis index when compared to docetaxel or 5-FU and CQ alone (Figures 6D-H). Concerning breast xenografts, the inhibition of tumor growth in mice treated with a combined therapy is correlated with a drastic increase in the proportion of cells that underwent apoptosis, as revealed by TUNEL staining (Figure S6). We have used fresh human tumor samples from prostate in an ex vivo slices tumor assay to confirm the efficiency of autophagy inhibition on ASP-8ADT anti-tumor activity alone or combined with conventional chemotherapy. This ex vivo tumor assay is an excellent tool as it keeps tumor heterogeneity and allows the clinical response to anticancer drugs to be predicted [START_REF] Majumder | Predicting clinical response to anticancer drugs using an ex vivo platform that captures tumour heterogeneity[END_REF].

In these experiments, human prostate tumors were treated with a complete panel of treatments. First, we confirmed the efficiency of autophagy inhibition on ASP-8ADT anti-tumor activity with an important increase in the proportion of cells undergoing apoptosis, as re-vealed by TUNEL staining (Figure 6I). Second, the combined therapy (ASP-8ADT/CQ) significantly increased the capacity of docetaxel/CQ to induce apoptosis (Figure 6I). Altogether, these assays clearly showed that inhibition of autophagy rescues the anti-cancer activity of ASP-8ADT and that a combined therapy based on a Ca 2+ stress and inhibition of autophagy increases chemosensitivity to docetaxel and 5-FU of prostate and breast cancer, respectively.

Discussion

In a previous work, we uncovered an atypical mode to escape from cell-death in response to Ca 2+ homeostasis perturbations [START_REF] Dubois | The calcium-signaling toolkit: Up-dates needed[END_REF][START_REF] Dubois | Differential effects of thapsigargin analogues on apoptosis of prostate cancer cells: complex regulation by intracellular calcium[END_REF] but the precise molecular mechanisms were not investigated. Here we identified autophagy and mitochondrial fission as gatekeepers in the paradigm referred as the Ca 2+ apoptosis link [START_REF] Berridge | The versatility and universality of calcium signalling[END_REF]. This dependency has never been identified before as a required step. Our most important finding is also that Ca 2+ -induced MPTP opening cannot be longer considered as a potent apoptosis activator just by itself. Indeed, one of our most striking results is that while TG and its analogue ASP-8ADT induce similar ER, cytosolic and mitochondria Ca 2+ dynamics leading to MPTP opening, apoptosis induced by ASP-8ADT is very modest (Figures 1,2). We show that this "loss of function" is linked to its inability to block autophagy as it has been described for TG [START_REF] Engedal | Modulation of intracellular calcium homeostasis blocks autophagosome formation[END_REF][START_REF] Ganley | Distinct autophagosomallysosomal fusion mechanism revealed by thapsigargin-induced autophagy arrest[END_REF][START_REF] Saetre | Novel steps in the autophagic-lysosomal pathway[END_REF], thereby revealing a direct and crucial role of autophagy in the Ca 2+ apoptosis link (Figure 3). This feature may be supported by the fact that TG and ASP-8ADT, have similar but not identical chemical structure [START_REF] Dubois | Differential effects of thapsigargin analogues on apoptosis of prostate cancer cells: complex regulation by intracellular calcium[END_REF]. Originally it has been suggested that the inhibitory effect of TG on the autophagic flux, was independent of a functional ER stress response, but could be related to the activity of TG to inhibit SERCA and thus to influence downstream Ca 2+ signaling pathways involved in the regulation of autophagy [START_REF] Ganley | Distinct autophagosomallysosomal fusion mechanism revealed by thapsigargin-induced autophagy arrest[END_REF]. Our results are not in accordance with this possibility since ASP-8ADT induces both similar ER stress response and remodeling of Ca 2+ homeostasis compared to TG (Figure 1). This implies that TG targets an unidentified molecular mechanism preferentially implicated in the closure of autophagosomes as it has been suggested in a previous study [START_REF] Engedal | Modulation of intracellular calcium homeostasis blocks autophagosome formation[END_REF]. Nonetheless, we were able to restore the pro-apoptotic potency of ASP-8ADT by using pharmacological or genetic inhibition of autophagy.

We also demonstrated that both TG and ASP-8ADT induced potent mitochondrial Ca 2+ overload that causes MPTP opening. However, we observed that mitochondria are still able to retain large amounts of Ca 2+ and surprisingly, that the mitochondrial membrane potential could be preserved. These findings seem to be in contradiction with the fact that when MPTP is open, both Ca 2+ and proton gradients across the inner mitochondrial membrane are dissipated. In fact, we identified that a situation with totally depleted mitochondria from Ca 2+ can occur only when mitochondrial potential is totally collapsed by using a protonophore such as FCCP (Figure 4). Indeed, we observed that TG induced only partial mitochondrial depolarization compared to FCCP and we suggest that this feature could explain the retention of Ca 2+ . In fact, our results are in accordance with some studies in that the process of MPTP opening at the cellular level does not obey to all-or-none law as it has been suggested in other models [START_REF] Dumas | Effect of transient and permanent permeability transition pore opening on NAD(P)H localization in intact cells[END_REF]. Concerning ASP-8ADT, the mitochondrial membrane potential was virtually not affected despite the opening of MPTP suggesting that other mechanisms can potentially play a crucial role for maintaining mitochondrial membrane potential. In particular, we identified autophagy and mitochondrial dynamics as two crucial steps involved in the maintain of mitochondrial potential (Figures 3,4). Interestingly, we demonstrated that the pro-survival action of autophagy or mitochondrial dynamics could be used sequentially by the cells to try to overcome the stress mediated by mitochondrial Ca 2+ overload (Figures 4). Indeed, in the case of autophagy impairment, cells switch from the pro-survival autophagy pathway to a DRP-1 mediated mitochondrial dynamics to try to overcome the stress mediated by mitochondrial Ca 2+ overload, highlighting the role of DRP1 for maintaining mitochondrial potential as observed in other study [START_REF] Choi | Drp1-mediated mitochondrial dynamics and survival of developing chick motoneurons during the period of normal programmed cell death[END_REF]. In this context, our results clearly indicated that the pharmacological or genetic impairment of DRP-1 could be used to restore the Ca 2+ apoptosis link. Thus, our study highlights the central role of autophagy and mitochondrial dynamics as survival mechanisms ensuring the integrity of mitochondrial membrane potential during mitochondrial Ca 2+ overload stress. For this reason, the targeting of autophagy or mitochondrial dynamics could be used to promote apoptosis in cancer cells subjected to mitochondrial Ca 2+ overload.

Our preclinical data confirmed that the use of Chloroquine or Rab7 GTPase inhibitor by inhibiting autophagy, increase chemotherapy efficiency in several cancer model in vitro.

As a proof of concept, we confirmed the potential translational significance of our combined strategy with conventional drugs used in clinic or in clinical evaluation (i.e. ASP-8ADT). For this, we used both classical in vivo approach (xenograft mouse model) and an ex-vivo strategy with human clinical specimens of prostate and breast cancer (Figures 6,S6). This ex-vivo strategy allows the prediction of the clinical response to anticancer drugs by maintaining tumor heterogeneity [START_REF] Majumder | Predicting clinical response to anticancer drugs using an ex vivo platform that captures tumour heterogeneity[END_REF]. In the case of prostate cancer treatment, we have used docetaxel which was the first chemotherapeutic agent to increase survival time in patients with androgen-resistant prostate cancer. For breast cancer we have evaluated the conventional treatment 5-FU. The ASP-8ADT compound used in our study to induce sustained mitochondrial perturbations corresponds to the active ingredient of the Mipsagargin pro-drug (G-202) [START_REF] Denmeade | Engineering a prostate-specific membrane antigen-activated tumor endothelial cell prodrug for cancer therapy[END_REF]) currently under evaluation as potential targeted therapy for prostate cancer, gliomas, clear cell renal cancer, or hepato-carcinomas. In a clinical study on patients with advanced solid tumors receiving Mipsagargin, authors did not observed a clinical response, but a prolonged disease stabilization was observed in a subset of patients [START_REF] Mahalingam | Mipsagargin, a novel thapsigargin-based PSMA-activated prodrug: results of a first-in-man phase I clinical trial in patients with refractory, advanced or metastatic solid tumours[END_REF]. In this context, our findings may benefit to patients by improving the clinical outcome associated with G-202 treatment. Indeed, our fundamental findings redefine the molecular pathways underlying the anti-tumor activity of ASP-8ADT (G-202) as we clearly demonstrated that concomitant inhibition of autophagy is required. In agreement with these findings, we proposed two new combined anticancer therapies (1) G-202 combined with CQ (2) G-202 combined with CQ and a standard chemotherapy. Concerning the combination with a standard chemotherapy, we clearly demonstrated that the combination of ASP-8ADT and docetaxel in the background of autophagy inhibition shown synergistic interactions (Figure 6). It is also well known that mitochondrial priming mediated by BCL-2 family proteins correlates with clinical response to cytotoxic chemotherapy [START_REF] Ni Chonghaile | Pretreatment mitochondrial priming correlates with clinical response to cytotoxic chemotherapy[END_REF]. Indeed, permeabilization of the OMM is considered to be the decisive event in the onset of cell death [START_REF] Kroemer | Mitochondrial membrane permeabilization in cell death[END_REF] and is subject to a tight control by pro-and anti-apoptotic members of the BCL-2 protein family [START_REF] Czabotar | Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy[END_REF]. Interestingly, the mechanism identified here for the sensitivity and the resistance to cancer therapies seems to be a BCL-2 independent mechanism and thus, it may represent an innovative therapeutic approach. Importantly, all the compounds used in our proposed combination therapies have been approved by the FDA. All compounds used in our combined therapies have also distinct mechanisms of antitumor activity ruling out a possible competitive impairment and both CQ and G-202 have already demonstrated an acceptable tolerability [START_REF] Manic | Chloroquine and hydroxychloroquine for cancer therapy[END_REF]. Such combined therapy using a targeted anticancer agent such as Mipsagargin in combination with a standard chemotherapy and inhibition of autophagy is a realistic approach. Recently Inspyr Therapeutics (G-202) have initiated a pre-clinical study evaluating the potential of G-202 in combination with Nexavar, using liver tumor models that express PSMA (Prostate Specific Membrane Antigen), the target of G-202. Moreover, numerous clinical studies also evaluate combined therapies including 2, 3 or 4 compounds. In particular, the combination of oxaliplatin, irinotecan, and 5fluorouracil (5-FU) (FOLFOXIRI) in association with bevacizumab demonstrated in a phase III trial to improve outcome and became a new standard regimen for initial therapy of metastatic colorectal cancer [START_REF] Loupakis | Initial therapy with FOLFOXIRI and bevacizumab for metastatic colorectal cancer[END_REF].

In conclusion, these findings challenge a crucial paradigm in cell death revealing a novel and promising priming strategy to improve clinical outcomes for patients with solid cancers.

Limitations of the Study

Based on the results derived from pharmacological induction of mitochondrial Ca 2+ overload in cultured cells and xenograft mouse models, we were able to demonstrate that, by inhibiting the autophagy pathway, mitochondrial Ca 2+ status can modulate chemosensitivity.

However, the conclusions would be strengthened if the precise molecular mechanism underlying autophagy inhibition and mediated by TG was identified. Moreover, the physiological and physiopathological aspects of this new interplay between Ca 2+ signaling and autophagy in the regulation of mitochondrial cell fate should be confirmed in the context of pathologies associated with mitochondrial disorders (neurodegenerative and cardiovascular diseases, diabetes or myopathies).

Highlights:

Mitochondrial Ca 2+ overload stimulus alone is ineffective to induce cell-death Autophagy is a gatekeeper of cell fate in response to Ca 2+ homeostasis disruption Mitochondrial Ca 2+ overload is a determinant of autophagy as a chemosensitizer

Figure 1 :

 1 Figure 1: Redefining the Ca 2+ -Apoptosis Link. (A) Representative measurements of SOCE

Figure 2 :

 2 Figure 2: Ca 2+ homeostasis disruption and MPTP opening. (A) MPTP opening was assessed directly by the calcein/Co 2+ method and calcein fluorescence quenching was imaged

Figure 3 :

 3 Figure 3: Differential effects of thapsigargin analogues on autophagy. (A) Representative

Figure 4 :

 4 Figure 4: Inhibition of autophagy determines the pro-apoptotic potential of mitochondrial Ca 2+ overload stimulus by allowing mitochondrial fission and membrane

Figure 5 :

 5 Figure 5: A co-targeting strategy based on mitochondrial Ca 2+ overload and autophagy

Figure 6 :

 6 Figure 6: Priming cancer cells by a concomitant inhibition of autophagy and Ca 2+ stress

  Photon intensity (A.U.)

Acknowledgments

We acknowledge Prof R. Tsien for generously providing 4mtTM3cpv Cameleon construct, and Prof J. Vuust Møller & Prof B. Christensen for providing thapsigargin analogues. Graphical Absract was adapted from Servier Medical Art, licensed under a Creative Common Attribution 3.0 Generic License. http://smart.servier.com/. This work was supported by a grant from Comité Septentrion de la Ligue contre le cancer to FV and La Fondation de France to CD.

Resource Availability

Lead contact fabien.vanden-abeele@inserm.fr

Materials Availability

Further information required to interpret, replicate or build upon the methods or findings reported in the manuscript is available from the corresponding author upon request (Fabien.vanden-abeele@inserm.fr).

Data and Code Availability

There is no dataset and/or code associated with the paper.

Methods

Authors Contributions

CD designed research studies, performed most of the experiments, conceived the experimental designs, analyzed the data and drafted the manuscript. AK, LN and GB performed experiments and analyzed the data. EV, FV and RT performed experiments. MR provided primary human stromal prostate cancer cells. DT participated to in vivo and ex-vivo experiments. JB provided primary human prostate cancer cells. NP provided advices. FV directed the study, designed research studies, performed the experiments, conceived the experimental designs, analyzed the data and drafted the manuscript.

CD and FV are corresponding authors: Fabien.vanden-abeele@inserm.fr; charlotte.dubois@inserm.fr

Declaration of Interests

The authors declare that they have no competing interests.

Ethics declarations

The present study was approved by Ethics Committee of GHICL Hospital, Hospital St-Philibert, Lomme, France.

Studies involving animals, including housing and care, method of euthanasia, and experimental protocols, were conducted in accordance with the local animal ethical committee in the animal house (C59-00913; protocol CEEA 202012) of the University of Sciences and Technologies of Lille, under the supervision of Dr. Lehen'kyi (59-009270).