Hexokinase 2 displacement from mitochondria‐associated membranes prompts Ca 2+ ‐dependent death of cancer cells
2020; Springer Nature; Volume: 21; Issue: 7 Linguagem: Inglês
10.15252/embr.201949117
ISSN1469-3178
AutoresFrancesco Ciscato, Riccardo Filadi, Ionica Masgras, Marco Pizzi, Oriano Marin, Nunzio Damiano, Paola Pizzo, Alessandro Gori, Federica Frezzato, Federica Chiara, Livio Trentin, Paolo Bernardi, Andrea Rasola,
Tópico(s)Pancreatic function and diabetes
ResumoReport8 May 2020Open Access Transparent process Hexokinase 2 displacement from mitochondria-associated membranes prompts Ca2+-dependent death of cancer cells Francesco Ciscato Francesco Ciscato Department of Biomedical Sciences (DSB), University of Padova, Padova, Italy Search for more papers by this author Riccardo Filadi Riccardo Filadi Department of Biomedical Sciences (DSB), University of Padova, Padova, Italy Search for more papers by this author Ionica Masgras Ionica Masgras Department of Biomedical Sciences (DSB), University of Padova, Padova, Italy Search for more papers by this author Marco Pizzi Marco Pizzi Surgical Pathology and Cytopathology Unit, Department of Medicine (DIMED), University of Padova, Padova, Italy Search for more papers by this author Oriano Marin Oriano Marin Department of Biomedical Sciences (DSB), University of Padova, Padova, Italy Search for more papers by this author Nunzio Damiano Nunzio Damiano Department of Biomedical Sciences (DSB), University of Padova, Padova, Italy Search for more papers by this author Paola Pizzo Paola Pizzo Department of Biomedical Sciences (DSB), University of Padova, Padova, Italy Search for more papers by this author Alessandro Gori Alessandro Gori CNR Institute of Chemistry of Molecular Recognition (ICRM), Milano, Italy Search for more papers by this author Federica Frezzato Federica Frezzato Hematology and Clinical Immunology Branch, Department of Medicine (DIMED), University of Padova, Padova, Italy Search for more papers by this author Federica Chiara Federica Chiara Department of Surgery, Oncology and Gastroenterology (DISCOG), University of Padova, Padova, Italy Search for more papers by this author Livio Trentin Livio Trentin Hematology and Clinical Immunology Branch, Department of Medicine (DIMED), University of Padova, Padova, Italy Search for more papers by this author Paolo Bernardi Paolo Bernardi orcid.org/0000-0001-9187-3736 Department of Biomedical Sciences (DSB), University of Padova, Padova, Italy Search for more papers by this author Andrea Rasola Corresponding Author Andrea Rasola [email protected] orcid.org/0000-0003-4522-3008 Department of Biomedical Sciences (DSB), University of Padova, Padova, Italy Search for more papers by this author Francesco Ciscato Francesco Ciscato Department of Biomedical Sciences (DSB), University of Padova, Padova, Italy Search for more papers by this author Riccardo Filadi Riccardo Filadi Department of Biomedical Sciences (DSB), University of Padova, Padova, Italy Search for more papers by this author Ionica Masgras Ionica Masgras Department of Biomedical Sciences (DSB), University of Padova, Padova, Italy Search for more papers by this author Marco Pizzi Marco Pizzi Surgical Pathology and Cytopathology Unit, Department of Medicine (DIMED), University of Padova, Padova, Italy Search for more papers by this author Oriano Marin Oriano Marin Department of Biomedical Sciences (DSB), University of Padova, Padova, Italy Search for more papers by this author Nunzio Damiano Nunzio Damiano Department of Biomedical Sciences (DSB), University of Padova, Padova, Italy Search for more papers by this author Paola Pizzo Paola Pizzo Department of Biomedical Sciences (DSB), University of Padova, Padova, Italy Search for more papers by this author Alessandro Gori Alessandro Gori CNR Institute of Chemistry of Molecular Recognition (ICRM), Milano, Italy Search for more papers by this author Federica Frezzato Federica Frezzato Hematology and Clinical Immunology Branch, Department of Medicine (DIMED), University of Padova, Padova, Italy Search for more papers by this author Federica Chiara Federica Chiara Department of Surgery, Oncology and Gastroenterology (DISCOG), University of Padova, Padova, Italy Search for more papers by this author Livio Trentin Livio Trentin Hematology and Clinical Immunology Branch, Department of Medicine (DIMED), University of Padova, Padova, Italy Search for more papers by this author Paolo Bernardi Paolo Bernardi orcid.org/0000-0001-9187-3736 Department of Biomedical Sciences (DSB), University of Padova, Padova, Italy Search for more papers by this author Andrea Rasola Corresponding Author Andrea Rasola [email protected] orcid.org/0000-0003-4522-3008 Department of Biomedical Sciences (DSB), University of Padova, Padova, Italy Search for more papers by this author Author Information Francesco Ciscato1, Riccardo Filadi1, Ionica Masgras1, Marco Pizzi2, Oriano Marin1, Nunzio Damiano1, Paola Pizzo1, Alessandro Gori3, Federica Frezzato4, Federica Chiara5, Livio Trentin4, Paolo Bernardi1 and Andrea Rasola *,1 1Department of Biomedical Sciences (DSB), University of Padova, Padova, Italy 2Surgical Pathology and Cytopathology Unit, Department of Medicine (DIMED), University of Padova, Padova, Italy 3CNR Institute of Chemistry of Molecular Recognition (ICRM), Milano, Italy 4Hematology and Clinical Immunology Branch, Department of Medicine (DIMED), University of Padova, Padova, Italy 5Department of Surgery, Oncology and Gastroenterology (DISCOG), University of Padova, Padova, Italy *Corresponding author. Tel: +39 049 8276062; E-mail: [email protected] EMBO Reports (2020)21:e49117https://doi.org/10.15252/embr.201949117 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Cancer cells undergo changes in metabolic and survival pathways that increase their malignancy. Isoform 2 of the glycolytic enzyme hexokinase (HK2) enhances both glucose metabolism and resistance to death stimuli in many neoplastic cell types. Here, we observe that HK2 locates at mitochondria-endoplasmic reticulum (ER) contact sites called MAMs (mitochondria-associated membranes). HK2 displacement from MAMs with a selective peptide triggers mitochondrial Ca2+ overload caused by Ca2+ release from ER via inositol-3-phosphate receptors (IP3Rs) and by Ca2+ entry through plasma membrane. This results in Ca2+-dependent calpain activation, mitochondrial depolarization and cell death. The HK2-targeting peptide causes massive death of chronic lymphocytic leukemia B cells freshly isolated from patients, and an actionable form of the peptide reduces growth of breast and colon cancer cells allografted in mice without noxious effects on healthy tissues. These results identify a signaling pathway primed by HK2 displacement from MAMs that can be activated as anti-neoplastic strategy. Synopsis Hexokinase 2 (HK2) localizes at mitochondria-associated membranes (MAMs) of tumor cells. A cell-penetrating peptide (HK2pep) dislodges HK2 from MAMs, prompts IP3R opening, mitochondria Ca2+ overload, calpain activation and tumor cell death. An activatable HK2pep inhibits tumor growth in vivo. In tumor cells, the key metabolic enzyme Hexokinase 2 (HK2) localizes at endoplasmic reticulum-mitochondria contact sites called MAMs (mitochondria-associated membranes). HK2 displacement from MAMs with a cell-penetrating peptide (HK2pep) elicits IP3R opening, mitochondrial Ca2+ overload and calpain activation, rapidly killing tumor cells. An activatable HK2pep is suitable for in vivo delivery and inhibits tumor growth without noxious effects on healthy tissues. Introduction Hexokinases are a family of four isoforms that catalyze phosphorylation of glucose, making it available for utilization in glycolysis, pentose phosphate pathway, glycogenesis, and hexosamine biosynthesis 1. HK2, the most active isozyme, is markedly expressed in cells characterized by a high rate of glucose consumption, such as adipose, skeletal, and cardiac muscle. During the neoplastic process, metabolic changes are required to allow cell growth in conditions of fluctuating nutrient and oxygen availability 2. HK2 plays a major role in this metabolic rewiring 3-5, being induced by oncogenic K-Ras activation 6 or in response to (pseudo)hypoxia 7, 8. HK2 is mainly bound to the outer mitochondrial membrane, where it can gain privileged access to newly synthesized ATP, thus increasing efficiency in glucose usage 9, while following glucose deprivation HK2 elicits autophagy by inhibiting mTORC1 10. HK2 binding to mitochondria is increased by Akt phosphorylation, a key metabolic event occurring downstream to many signaling pathways hyperactivated in tumor cells 11, and by interactions with DMPK and Src kinases 12, whereas it is inhibited by the Akt-antagonizing phosphatase PHLPP and by hexokinase enzymatic product glucose-6-phosphate 10. Moreover, mitochondrial HK2 takes part in the protection of cancer cells from noxious stimuli through poorly defined mechanisms that include antagonizing the activity of pro-apoptotic Bcl-2 family proteins and increasing anti-oxidant defenses through interaction with the fructose-2,6-bisphosphatase TIGAR, which elicits pentose phosphate pathway induction 13. In cancer patients, HK2 induction is related to stage progression, acquisition of invasive and metastatic capabilities, and poor prognosis 14. HK2 promotes neoplastic growth in glioblastoma multiforme 15, confers chemoresistance in epithelial ovarian cancer 16, and is required for tumor onset and maintenance in mouse models of lung and breast cancer, where its genetic ablation is therapeutic without adverse effects 6. Thus, HK2 constitutes a promising target for developing anti-neoplastic strategies, but the clinical use of hexokinase inhibitors is hampered by lack of specificity or side effects 17 potentially associated with glucose metabolism derangement. A possible alternative approach is detaching HK2 from mitochondria, as we and others have previously shown that this can induce opening of a mitochondrial channel, the permeability transition pore (PTP), and consequently cell death 12, 13, 18, 19. However, both a detailed comprehension of the molecular mechanisms leading to cell damage and the development of a HK2-targeting tool that is operational in in vivo tumor models are required to translate this information into the groundwork for future anti-neoplastic approaches. Here, we demonstrate that in neoplastic cells, HK2 localizes in MAMs, specific subdomains of interaction between mitochondria and ER. HK2 detachment from MAMs rapidly elicits a massive Ca2+ flux into mitochondria and consequently a calpain-dependent cell death. We ignite this process with a HK2-targeting peptide composed by modular units that can be adapted to in vivo delivery, without affecting hexokinase enzymatic activity and with no adverse effects on animal models. Results and Discussion HK2 localizes in MAMs of neoplastic cells Dissection of submitochondrial HK2 localization can provide important functional clues, as mitochondria compartmentalize specific activities in domains formed by multiprotein platforms. After confirming that HK2 associates with tumor cell mitochondria (Fig 1A), we have found that it specifically localizes in MAMs by merging the fluorescence of HK2-conjugated antibodies with mitochondria-targeted YFP and ER-targeted CFP (Fig 1B) or with a split-GFP-based probe for ER-mitochondria contacts (SPLICSL) 20 (Fig 1C). These experiments have been extended to diverse HK2-expressing tumor cell models (Fig EV1A and B), and their quantification indicate both that 70–80% of HK2 localizes in MAMs and that most cellular MAMs harbor HK2 proteins (Fig 1D–F). Interestingly, the use of a short-range, split-GFP-based approach (SPLICSS) 20 designed to identify proteins localized in the tighter MAM fraction does not detect HK2 (Fig EV1C). The SPLICSL analysis also showed that HK2 is significantly enriched in MAMs with respect to TOM20, a protein that is uniformly distributed in the outer mitochondrial membrane (Fig EV1D). MAMs are dynamic structures that control the exchange between ER and mitochondria of ions and lipids, tuning complex biological processes such as ER stress, autophagy, cell death and maintenance of glucose homeostasis 21-23. A pivotal role of MAMs is the regulation of Ca2+ fluxes from ER to mitochondria through IP3Rs 24; thus, HK2 displacement from MAMs could affect intracellular Ca2+ dynamics, raising the possibility that a Ca2+ dyshomeostasis can ensue and damage neoplastic cells. Figure 1. HK2 locates in MAM of cancer cells and is displaced by HK2pep A. Immunofluorescence staining of HK2 with an AlexaFluor488-conjugated antibody in HeLa cells expressing mitochondria-targeted RFP. Yellow signals in the merge analysis indicate mitochondrial localization of HK2. Scale bar: 15 μm. B. Immunofluorescence staining of HK2 with a secondary AlexaFluor555-conjugated antibody in HeLa cells expressing both mitochondria-targeted YFP and ER-targeted CFP. The merged white signal indicates MAM localization of HK2 and is quantified in the bar graph on the right (n = 24). Image magnifications are shown in the lower part of the panel; arrows indicate HK2 dots in mito-ER contact sites. Scale bar: 15 μm. C. Fluorescence co-staining of HK2 and split-GFP-based probe for ER-mitochondria contacts (SPLICSL) on HeLa cells; HK2 is revealed with a secondary AlexaFluor555-conjugated antibody, and the merged signal is white. Scale bar: 15 μm. D. Quantification of panel B experiment showing the percentage of HK2 dots that merge with MAMs in HeLa cells (n = 24 cells analyzed from three independent experiments; mean ± SD). E. Quantification of panel C experiment showing the percentage of SPLICSL dots positive for HK2 in HeLa cells (n = 10 cells analyzed from three independent experiments; mean ± SD). F. Percentage of HK2 dots positive for SPLICSL in HeLa, COLO 741, MDA-MB-231, PN 04.4, and S462 cells (n ≥ 6 cells for each cell line; mean ± SD). G. Functional unit composition of HK2pep (left); the HK2-targeting sequence is in red; the polycation and polyanion stretches are in light blue and light green, respectively; the MMP2/9 target sequence is in yellow. On the right, mass spectrometry profile of HK2pep before and after incubation (cl-HK2pep) with human MMP9. H. HK2 displacement from HeLa MAMs after a 2 min treatment with cl-HK2pep is shown by loss of merging signal analyzed as in (C). cl-SCRpep is used as a negative control, and Pearson's co-localization coefficient is indicated in figure (cl-SCRpep n = 26 cells; cl-HK2pep n = 24 cells; P < 0.001 with a Student's t-test). Scale bar: 10 μm. I, J. Effect of cl-HK2pep on glucose phosphorylation by human recombinant HK2 (I) or in 4T1 cell extracts (J), where both total hexokinase activity and HK1/HK2 specific activities are measured. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Related to Fig 1. HK2 locates in MAMs of cancer cells Western immunoblot analysis of HK2 expression on neoplastic cells of human origin (cervix carcinoma HeLa cells, colorectal carcinoma COLO 741 cells, breast cancer MDA-MB-231 cells, plexiform neurofibroma PN 04.4 cells, malignant peripheral nerve sheath tumor S462 cells, B-chronic lymphocytic leukemia MEC1 cells) and of mouse origin (colon carcinoma CT26 cells and breast cancer 4T1 cells). ATP5A and GAPDH are mitochondrial and cytosolic loading controls, respectively. Fluorescence co-staining of HK2 and split-GFP-based probe for ER-mitochondria contacts (SPLICSL) on colorectal carcinoma COLO 741 cells, breast cancer MDA-MB-231 cells, plexiform neurofibroma PN 04.4 cells, and malignant peripheral nerve sheath tumor S462 cells. HK2 is revealed with an AlexaFluor555-conjugated secondary antibody. Scale bar, 15 μm. Fluorescence co-staining of HK2 and split-GFP-based probe for ER-mitochondria contacts SPLICSS. HK2 co-localization with SPLICSL (related to Fig 1H) or SPLICSS is quantified in the bar graph on the right (mean ± SEM obtained from at least three independent experiments). Scale bar, 20 μm. Both HK2 and the outer mitochondrial membrane marker TOM20 are analyzed for their co-localization with MAMs. HK2 is revealed with an AlexaFluor555-conjugated secondary antibody and TOM20 with an AlexaFluor647-conjugated secondary antibody; the merged signal is white in both analyses. MAM localization of HK2 or TOM20 is quantified in the bar graph on the right (mean ± SEM obtained from three independent experiments). Scale bar, 15 μm. Download figure Download PowerPoint Design of a peptide that displaces HK2 from MAMs without affecting hexokinase enzymatic activity To investigate this possibility and to generate a tool with a potential anti-tumor activity in vivo, we have conceived a HK2-targeting cell penetrating peptide (CPP), dubbed HK2pep (Fig 1G). HK2pep is designed with a modular structure composed of: (i) a N-terminal HK2 tail, which acts as the active moiety by displacing HK2 from the outer mitochondrial membrane (in the negative control, SCRpep, this HK2-specific sequence is substituted by a scrambled one); (ii) a polycation stretch required for plasma membrane permeation; (iii) a polyanion sequence that shields polycation charges; and iv) a matrix metalloprotease 2 and 9 (MMP2/9) target sequence that links the two charged stretches. As previously observed with similar actionable CPPs 25, the metalloprotease target sequence inhibits cell uptake of the peptide until its polycation sequence is unmasked by MMP2/9 cleavage. MMPs are highly expressed in a variety of tumor types, where they induce extracellular matrix remodeling and favor cancer cell invasiveness 26. Hence, HK2pep should be preferentially activated inside neoplasms, and its subsequent entry through the plasma membrane would then lead to HK2 displacement from mitochondria and eventually cancer cell death. Moreover, HK2pep is not permeable across the endothelium of normal blood vessels, but its dimension (about 5 kDa) allows passage across the fenestrated endothelium that perfuses many cancer types. The active moiety of HK2pep (i.e., the cleaved peptide, cl-HK2pep, Fig 1G) is indeed able to enter cells, to interact with mitochondria (Fig EV2A), and to induce HK2 detachment from MAMs in < 2 min (Figs 1H and EV2B). cl-HK2pep does not perturb hexokinase enzymatic activity either on the purified enzyme (Fig 1I) or on cell samples, where it is equally ineffective on HK2 and on the widespread isozyme HK1 (Figs 1J and EV2C). Moreover, the use of a subtoxic peptide concentration indicates that it does not affect the glycolytic activity of the target cell (Fig EV2D). Click here to expand this figure. Figure EV2. Related to Fig 1. Characterization of cl-HK2pep HeLa cells expressing mitochondrial-targeted RFP are treated for 2 min with either cl-SCRpep or cl-HK2pep (1 μM each) labeled with the green fluorophore ATTO 488. The yellow signal indicates mitochondrial localization of the peptide. Scale bar: 15 μm. HK2 displacement from HeLa mitochondria after a 2-min treatment with cl-HK2pep is shown by loss of merging signal analyzed as in Fig 1C. cl-SCRpep is used as a negative control, and Pearson's co-localization coefficient is indicated in figure (cl-SCRpep n = 40 cells; cl-HK2pep n = 51 cells; P < 0.01 with Student's t-test). Scale bar: 15 μm. Effect of 10 μM cl-HK2pep on glucose phosphorylation in CT26 cell extracts, where both total hexokinase activity and HK1/HK2-specific activities are measured (mean ± SD, n = 3 independent experiments). Extracellular acidification rate measurements performed on HeLa cells treated with either cl-SCRpep or cl-HK2pep (200 nM; mean ± SEM, n = 3 independent experiments, Student's t-test analysis n.s.). Download figure Download PowerPoint HK2 detachment from MAMs elicits a Ca2+ flux into mitochondria via IP3Rs and plasma membrane that causes mitochondrial depolarization In accord with a role played by several MAM proteins in the regulation of Ca2+ homeostasis, cl-HK2pep prompts cycles of ER Ca2+ release and refill (Fig 2A) and boosts cytosolic IP3 levels (Fig 2B). This IP3 rise is prevented by pre-incubation with the IP3R inhibitor Xestospongin C (Xe-C; Fig 2B) and by chelating cytosolic Ca2+ (Fig EV3A), and delayed with respect to the Ca2+ release from ER (compare Fig 2A and B). These observations are consistent with cl-HK2pep eliciting a primary Ca2+ efflux from ER that prompts a surge in cytosolic IP3 27. Indeed, Ca2+ enhances PLC activity that generates IP3 28, further amplifying ER Ca2+ release via IP3Rs. Figure 2. HK2 detachment from MAMs prompts Ca2+ influx and depolarization in mitochondria following IP3R opening A–F. Effect of cl-HK2pep on cellular Ca2+ dynamics and IP3 levels. ER Ca2+ levels are measured by the FRET-based, D4ER fluorescent probe expressed in the lumen of ER (A); IP3 levels are assessed with the GFP-PHD probe; histamine (100 μM) is used as a positive control for IP3 generation; data are reported as mean of fluorescent signals ± SEM (n = 3 independent experiments; B); changes in mitochondrial Ca2+ levels are recorded (C; scale bar: 20 μm) and quantified using the GCAMP6f sensor (in D, as mean of 475/410 nm ratio; signal ± SEM of at least five independent experiments and more than 20 cells analyzed), in (E) as percentage of cells with increased Ca2+ in mitochondria, with a threshold 475/410 ratio for positivity > 3; baseline mean ratio = 1.84 ± 0.54) or with mitochondria-targeted aequorin (F, where data are reported as mean of [Ca2+] ± SEM of 3 independent experimental days). G–J. Effect of cl-HK2pep treatment on mitochondrial membrane potential assessed with the TMRM probe. Kinetic experiments (G, single cell analysis, n = 216 cells analyzed in at least 10 independent experiments; H, representative field; scale bar: 20 μm) are quantified (I and J; TMRM fluorescence is normalized to initial value and expressed in percentage for each time point, with a depolarization threshold placed at 40% of initial value). Data information: Experiments throughout the figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each). Where indicated, cells are kept in Ca2+-free medium plus 500 μM EGTA with or without 10 μM BAPTA-AM; Xe-C is Xestospongin C, which selectively inhibits IP3R at the 5 μM concentration used here 46. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Related to Fig 2. Kinetics of cl-HK2pep-induced mitochondrial depolarization A. IP3 levels are assessed with the GFP-PHD probe in presence or not of 10 μM BAPTA-AM; data are reported as mean of fluorescent signals ± SEM (n = 3 independent experiments) B–E. Changes in mitochondrial membrane potential following cl-HK2pep treatment assessed with TMRM as in Fig 2G. In B and C, HeLa cells are treated with 5 μM cl-HK2pep; mitochondrial depolarization is shown for single cells (B; n = 60 cells analyzed from three independent experimental days) or as an averaged trace (C; n = 60 cells analyzed from three independent experimental days). TMRM signal is normalized to the initial value and expressed in percentage for each time point. D, TMRM positive HeLa cells are expressed in percentage (threshold count: fluorescence signal > 40% of initial value; n = 60 cells analyzed from three independent experimental days). E, HeLa cells are treated with 2 μM cl-HK2pep (n = 216 cells analyzed from 10 independent experimental days; fluorescence mean ± SEM) and analyzed as in C. F, G. Changes in Ca2+ levels and membrane potential in HeLa cell mitochondria following treatment with 2 μM cl-HK2pep. Measurements are performed in parallel on cells expressing mito-GCAMP6f and loaded with TMRM. Two different single representative cell traces are shown, characterized by fast (F) or slow (G) mitochondrial depolarization following peptide treatment (three different experimental days). The dotted line indicates the time point in which mito-GCAMP6f ratio increases from the basal. H. Mitochondrial depolarization in 4T1 cells treated with 2 μM cl-HK2pep (n = 129) is analyzed as in C. Where indicated, BAPTA-AM (10 μM; n = 147) or Xe-C (5 μM; n = 141) are added as in Fig 2B 1 h before experiment. I. Co-immunoprecipitation between HK2 and GRP75. Download figure Download PowerPoint Mitochondria can promptly take up Ca2+ released from ER, resulting in modulation of the activity of Krebs cycle enzymes and preventing deregulated increases in cytosolic [Ca2+] 24, 29. cl-HK2pep treatment rapidly boosts mitochondrial [Ca2+] in a stable and Xe-C-sensitive way (Movie EV1 and Fig 2C–E), rising mitochondrial [Ca2+] to about 50 μM (Fig 2F). However, chelation of extracellular Ca2+ only induces a transient mitochondrial [Ca2+] peak of about 12 μM (Fig 2F), indicating that cl-HK2pep administration elicits both Ca2+ release from ER and Ca2+ entry through plasma membrane, probably as a secondary effect 30, and that mitochondria take up Ca2+ from both sources. HK2 targeting also prompts a sudden and massive mitochondrial depolarization (Fig 2G and H, Movies EV2 and EV3 and Fig EV3B–E), which follows the increase in mitochondrial [Ca2+] (Fig EV3F and G). This depolarization is inhibited by chelating cytosolic or extracellular Ca2+ and by the IP3R inhibitor Xe-C (Fig 2I and J, Movie EV4 and Fig EV3H). Therefore, the HK2-targeted peptide causes mitochondrial Ca2+ overload as a consequence of Ca2+ release form ER via IP3Rs and of Ca2+ entry through plasma membrane. This signaling is further supported by the observation that HK2 co-immunoprecipitates with GRP75 (Fig EV3I), a chaperone that directly interacts with IP3R at MAMs and favors mitochondrial Ca2+ uptake upon IP3-dependent Ca2+ release from the ER. This interaction is in line with the observation that HK2 locates to loose mitochondria-ER contacts sites (Fig 1C and F), which can accommodate the bulky IP3R-Grp75-VDAC complexes 24. The HK2-targeting peptide triggers calpain-dependent cell death Overcoming the efflux and the buffering capacity of Ca2+ in mitochondria can induce the permeability transition pore (PTP), a high conductance channel the opening of which commits cells to death 31, 32. PTP opening is independently inhibited by two unrelated molecules, Cyclosporin-A (CsA) and C63, but this inhibitory effect can be overwhelmed by intense stimuli of PTP induction 33. We find that both CsA and C63 do not affect mitochondrial Ca2+ uptake following cl-HK2pep treatment (Fig 3A), but markedly delay mitochondrial depolarization (Fig 3B and Movies EV5 and EV6), indicating that this occurs downstream to PTP induction. Figure 3. HK2pep induces PTP- and calpain-dependent cell death A, B. Effects of the PTP desensitizers CsA or C63 (5 μM each, 1 h pre-incubation) on mitochondrial Ca2+ levels recorded with GCAMP6f (A) and on mitochondrial membrane potential assessed with TMRM (B) in cells treated with cl-HK2pep. C–F. Cell death induction by cl-HK2pep; in the cytofluorimetric analyses reported in C, E, and F, viable cells are double negative for Annexin V-FITC and 7-AAD staining and measured 15 min after peptide treatment; in the kinetic experiment shown in D (scale bar: 50 μm), viable cells are double negative for Annexin V-FITC and 7-AAD and TMRM positive. In C and F, data are presented as mean ± SD and they were obtained from 3 different experiments or more; 3 technical replicates in all analyzed experiment. G, H. Effect of calpain inhibition on mitochondrial membrane potential assessed with TMRM (G, normalized fluorescence signal mean ± SD; three different experiments including three technical replicates each) and on mitochondrial Ca2+ levels recorded with GCAMP6f (H) in cells treated with cl-HK2pep. Data information: Experiments throughout the figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each), where indicated, the caspase inhibitor Z-VAD-fmk or the calpain inhibitor PD150606 (50 μM each) are pre-incubated 1 h before peptide treatment. Experiments using TMRM or GCAMP6f probes are analyzed as in Fig 2. In C, F, and G, data are presented as mean ± SD of at least three independent experiments. In C ***P < 0.001 with Student's t-test; in F and G, P < 0.0001 with a two-way ANOVA (cl-HK2pep treatment versus cl-HK2pep+PD150606 or cl-SCRpep treatments); Bonferroni post-test in graph ***P < 0.001 (cl-HK2pep versus cl-SCRpep and cl-HK2pep versus PD150606 + cl-HK2pep). Download figure Download PowerPoint The HK2-targeting peptide abruptly elicits cell death in all tested cancer cell models (Fig 3C). The effect of cl-HK2pep did not change when cells were kept in the absence of glucose or were treated with HK inhibitors (Fig EV4A and B), indicating that it is independent of HK2 enzymatic activity. Peptide administration triggers mitochondrial depolarization in most tumor cells after 2–4 min, followed by phosphatidylserine exposure on the cell surface and plasma membrane rupture in < 1 h (Fig 3D and E, Movie EV7). Even though these are typical apoptotic changes, none of them is affected by the pan-caspase inhibitor Z-VAD-fmk (Figs 3E and EV4C–E). However, the broad-spectrum calpain inhibitor PD150606 abrogates cl-HK2pep-dependent induction of mitochondrial depolarization and cell death (Figs 3E–G and EV4F–L) without affecting the mitochondrial Ca2+ rise triggered by the peptide (Fig 3H). Taken together, these data indicate that HK2 displacement from MAMs does not trigger a classical apoptotic pathway, but rather activates a cell death process relying on the Ca2+-dependent proteases calpains 34. We also evaluated the effect of cl-HK2pep administration on non-transformed cell types. Mouse RAW 264.7 macrophages and mouse C2C12 myoblasts express HK2 (Fig EV4M), even though it is barely located in MAMs (Fig EV4N). Treatment with cl-HK2pep is poorly effective in inducing death of these cell models (Fig EV4O a
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