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DRP-1 functions independently of mitochondrial structural perturbations to facilitate BH3 mimetic-mediated apoptosis

DRP-1 functions independently of mitochondrial structural perturbations to facilitate BH3... Maintenance of mitochondrial integrity is critical for normal cellular homoeostasis. Most cells respond to stress stimuli and undergo apoptosis by perturbing mitochondrial structure and function to release proteins, such as cytochrome c, which are essential for the execution of the intrinsic apoptotic cascade. Cancer cells evade these events by overexpressing the anti-apoptotic BCL-2 family of proteins on mitochondrial membranes. Inhibitors of the anti- apoptotic BCL-2 family proteins, also known as BH3 mimetics, antagonise the pro-survival functions of these proteins and result in rapid apoptosis. Although the precise mechanism by which BH3 mimetics induce apoptosis has been well characterised, not much is known in terms of the structural changes that occur in mitochondria during apoptosis. Using a panel of highly selective BH3 mimetics and a wide range of cell lines, we demonstrate that BH3 mimetics induce extensive mitochondrial fission, accompanied by swelling of the mitochondrial matrix and rupture of the outer mitochondrial membrane. These changes occur in a BAX/ BAK-dependent manner. Although a major mitochondrial fission GTPase, DRP-1, has been implicated in mitochondrial apoptosis, our data demonstrate that DRP-1 might function independently/downstream of BH3 mimetic-mediated mitochondrial fission to facilitate the release of cytochrome c and apoptosis. Moreover, downregulation of DRP-1 prevented cytochrome c release and apoptosis even when OPA1, a protein mediating mitochondrial fusion, was silenced. Although BH3 mimetic-mediated displacement of BAK and other BH3-only proteins from BCL-X and MCL-1 was unaffected by DRP-1 downregulation, it prevented BAK activation significantly, thus placing DRP-1 as one of the most critical players, along with BAX and BAK, that governs BH3 mimetic-mediated cytochrome c release and apoptosis. Introduction mitochondrial membrane (IMM) and formation of the Most chemotherapeutic agents kill cancer cells by apoptosome that activates the initiator and effector cas- executing the intrinsic apoptotic pathway, which is char- pases. MOMP is regulated by the BCL-2 family, whereby acterised by mitochondrial outer membrane permeabili- BAX and BAK, undergo specific conformational changes zation (MOMP), release of cytochrome c from the inner to form oligomeric pores that insert into the outer mitochondrial membrane (OMM) to release cytochrome 1,2 c . Activation of BAX and BAK is achieved by several Correspondence: Shankar Varadarajan (svar@liv.ac.uk) pro-apoptotic BH3-only members, which are generally Department of Molecular and Clinical Cancer Medicine, Institute of rendered ineffective by sequestration with specific anti- Translational Medicine, University of Liverpool, Liverpool, Ashton Street, apoptotic BCL-2 family of proteins, such as BCL-2, BCL- Liverpool L69 3GE, UK 2 3,4 Department of Cellular and Molecular Physiology, Institute of Translational X and MCL-1 . These anti-apoptotic proteins are Medicine, University of Liverpool, Liverpool, Ashton Street, Liverpool L69 3GE, highly expressed in many cancers and inhibitors known as UK BH3 mimetics have been designed to target them in order Full list of author information is available at the end of the article. Edited by I. Amelio © 2019 The Author(s). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Milani et al. Cell Death Discovery (2019) 5:117 Page 2 of 11 to displace the BH3-only proteins, activate BAX and BAK, Results thereby inducing MOMP and apoptosis of cancer cells . BH3 mimetics induce marked mitochondrial structural ABT-737, and its orally available analogue, ABT-263 changes (Navitoclax) were the first bona fide BH3 mimetics Previously, we have reported that BH3 mimetics induce 6,7 developed to target BCL-2, BCL-X and BCL-w . Sub- a novel paradigm of apoptosis characterised by marked sequently, BH3 mimetics that specifically target BCL-2 ultrastructural changes in the mitochondria, involving the (ABT-199 (Venetoclax), S55746), BCL-X (A-1331852) loss of mitochondrial cristae and the appearance of breaks and MCL-1 (A-1210477, S63845, AMG 176 and in the OMM, resulting from mitochondrial matrix swel- 8–14 19,20,31 AZD5991) have been synthesised . These inhibitors, as ling . In cell lines that depend for survival almost single agents, have demonstrated much promise in treat- exclusively on BCL-2 (MAVER-1), BCL-X -(K562) and ing a wide variety of haematological malignancies, and MCL-1 (H929) , exposure to the relevant BH3 mimetics, have had limited success in combination with conven- such as ABT-199, A-1331852 and A-1210477, respec- 8,12–17 tional chemotherapy against several solid tumours . tively, resulted in similar mitochondrial matrix swelling BH3 mimetics induce apoptosis primarily by targeting and rupture of the OMM (Fig. 1a–c). Such mitochondrial protein–protein interactions between the anti- and pro- changes were also evident in H1299 cells following apoptotic BCL-2 family members . Subsequently, BH3 exposure to a combination of A-1331852 and A-1210477, mimetics have been shown to induce significant structural as these cells depend on both BCL-X and MCL-1 for changes in the mitochondria, ranging from mitochondrial survival (Fig. 1d). These mitochondrial ultrastructural matrix swelling to discontinuities in the OMM, upstream changes were independent of effector caspases, as they 19,20 of caspase activation . Furthermore, BAX and BAK were observed in cells pre-treated with Z-VAD.fmk, a localise to the breakpoints in OMM and may facilitate broad-spectrum caspase inhibitor (Fig. 1). Exposure of the cytochrome c release at such breakpoints . Although different cells to their appropriate BH3 mimetic resulted BCL-2 family members have been implicated in regulating in mitochondrial membrane depolarisation, loss of cyto- 21–24 mitochondrial membrane dynamics and functions , chrome c and induction of apoptosis, as assessed by putative inhibitors of MCL-1 have often resulted in phosphatidylserine externalisation (Supplementary Fig. 25–27 extensive mitochondrial fission in various cell lines . S1). Exposure of the cells to Z-VAD.fmk almost com- The regulation of this fission and its relationship to BH3 pletely inhibited BH3 mimetic-mediated apoptosis, mimetic-mediated apoptosis remains to be determined. assessed by PS externalisation, whereas little if any inhi- Mitochondrial structure is maintained through an bition of cytochrome c release was observed (Supple- intricate balance between the activities of several fusion mentary Fig. S1). Taken together these results suggested and fission proteins, which belong to a conserved family that the mitochondrial structural changes occurred of GTPases that reside in the OMM or IMM. Mitofusins 1 upstream of effector caspase activation and accompanied and 2 (MFN1/ 2) and optic atrophy 1 (OPA1) are essential cytochrome c release, as well as a loss of mitochondrial for mitochondrial fusion, whereas dynamin related pro- membrane potential. tein 1 (DRP-1) is essential for mitochondrial fission . Defects in mitochondrial fusion and fission have been BH3 mimetic-mediated mitochondrial perturbations occur implicated in a range of pathophysiological conditions in a BAX/BAK-dependent manner including poor brain development, optic atrophy, cardi- To assess whether BAX and BAK play crucial roles in 29,30 omyopathy and neurodegenerative diseases . Mount- BH3 mimetic-mediated ultrastructural changes in mito- ing evidence now suggests the involvement of several chondria, we exposed HCT-116 WT and BAX/BAK members of BCL-2 family members, particularly MCL-1, double knock-out (DKO) cells to a combination of A- in the regulation of mitochondrial structure and func- 1331852 and A-1210477, as HCT-116 cells also depend 22–25,29 33 tion . However, the mechanism by which MCL-1 on both BCL-X and MCL-1 for survival . Exposure of regulates mitochondrial membrane dynamics and the the HCT-116 WT cells to the BH3 mimetics resulted in potential cross-talk with its conventional role in antag- significant mitochondrial matrix swelling accompanied by onising apoptosis remain to be characterised. a loss of mitochondrial cristae, although rupture of the In this study, we use a panel of highly selective BH3 OMM was not readily apparent (Fig. 2). However, all mimetics together with cell lines that depend on specific these mitochondrial changes were clearly prevented in the BCL-2 family members for survival to demonstrate that HCT-116 BAX/BAK DKO cells, demonstrating a BH3 mimetics induce significant ultrastructural mito- requirement for BAX and/or BAK for the perturbation of chondrial changes upstream of caspase activation. DRP-1 the mitochondria. Since BH3-only members are generally plays a role downstream of these changes but upstream of required to activate BAX and BAK, we wished to assess MOMP to facilitate cytochrome c release and apoptosis, whether BH3 mimetics could induce mitochondrial following exposure to BH3 mimetics. structural perturbations in the absence of all known pro- Official journal of the Cell Death Differentiation Association Milani et al. Cell Death Discovery (2019) 5:117 Page 3 of 11 Fig. 1 BH3 mimetics induce marked ultrastructural changes in mitochondria of different cells. a MAVER-1, b K562, c H929 and d H1299 cells were exposed to Z-VAD.fmk (30 µM) for 0.5 h, followed by ABT-199 (100 nM), A-1331852 (100 nM), A-1210477 (10 µM), or a combination of A-1331852 (100 nM) and A-1210477 (10 µM), respectively, for 4 h and assessed for mitochondrial structural changes by electron microscopy. Yellow arrowheads indicate regions of breaks at the outer mitochondrial membrane. Scale bars: 500 nm HCT-116 OctaKO HCT-116 WT HCT-116 BAX/BAK DKO Fig. 2 BH3 mimetics disrupt mitochondria in a BAX- and BAK-dependent but BH3-independent manner. HCT-116 WT, DKO (BAX/BAK deficient) and OctaKO cells were exposed to Z-VAD.fmk (30 µM) for 0.5 h, followed by a combination of A-1331852 (100 nM) and A-1210477 (10 µM) for 4 h and assessed for mitochondrial structural changes by electron microscopy. Scale bars: 500 nm Official journal of the Cell Death Differentiation Association A-1210477+A-1331852 DMSO Milani et al. Cell Death Discovery (2019) 5:117 Page 4 of 11 DMSO A-1331852 A-1210477 A-1331852+A-1210477 S63845 A-1331852+S63845 DMSO A-1210477 S63845 (1 uM) S63845 (10 uM) Control siRNA DRP-1 siRNA *** *** Fig. 3 Mitochondrial fission mediated by A-1210477 and S63845 occurs in a DRP-1-dependent manner. a H1299 cells were exposed to Z- VAD.fmk (30 µM) for 0.5 h, followed by either A-1331852 (100 nM), A-1210477 (10 µM), S63845 (100 nM), or a combination of the different inhibitors for 4 h and assessed for mitochondrial integrity by immunostaining with HSP70 antibody. b H1299 cells were transfected with control or DRP-1 siRNA for 72 h and exposed to A-1210477 (10 µM) or S63845 (1 and 10 µM) for 4 h and assessed for mitochondrial integrity. The extent of mitochondrial fragmentation was quantified by analysing ~100 cells for each condition in three independent experiments. Scale bar: 10 µm. Error bars = mean ± SEM. Statistical analysis was conducted by one-way ANOVA (***P ≤ 0.001) apoptotic BH3-only members. For this, we used HCT-116 suggested that this could be a prerequisite for the ensuing 25–27 OctaKO cells, which lack the BH3-only members namely, apoptosis in MCL-1-dependent cell lines . In support BIM, BID, PUMA, BAD, BIK, HRK, BMF and NOXA . of this suggestion, exposure of A-1210477 but not A- Exposure of these cells to a combination of A-1331852 1331852 resulted in extensive mitochondrial fission that and A-1210477 resulted in mitochondrial structural resembled mitochondrial fragmentation in H1299 cells changes, characteristic of significant cristae remodelling (Fig. 3a, Supplementary Fig. S2). The ability of A-1210477 (Fig. 2). However, the swelling of mitochondrial matrix to induce mitochondrial fission was also clearly evident and the accompanying loss of cristae observed in the when used in combination with A-1331852 to induce HCT-116 WT cells following BH3 mimetics were not apoptosis in these cells (Fig. 3a, Supplementary Fig. S2). apparent in HCT-116 OctaKO cells (Fig. 2). This is con- However, mitochondria in this instance appeared swollen, sistent with earlier findings demonstrating that the pro- potentially indicating swollen matrix and loss of cristae apoptotic BH3-only members are dispensable for BH3 that were previously observed at the level of electron mimetic-mediated apoptosis . Taken together, our data microscopy (compare Figs. 1d and 3a). In marked con- demonstrated that the activation of BAX and/or BAK, trast, S63845 at a concentration (100 nM) sufficient to either in a BH3-dependent or independent manner, is induce apoptosis in a MCL-1-dependent manner failed essential for the ultrastructural changes observed in the to demonstrate mitochondrial fission (Fig. 3a, Supple- mitochondria, following exposure to BH3 mimetics. mentary Fig. S2). However, S63845 (100 nM) when used in conjunction with A-1331852 resulted in mitochondrial DRP-1 is not required for the mitochondrial structural structural changes that resembled the swollen mito- changes that occur during the onset of apoptosis chondria observed following a combination of A-1210477 We previously reported that putative inhibitors of and A-1331852 (Fig. 3a, Supplementary Fig. S2). Taken MCL-1 induced extensive mitochondrial fission and together, our results suggested that mitochondrial fission Official journal of the Cell Death Differentiation Association DMSO A-1210477 S63845 (1 uM) S63845 (10 uM) DRP-1 siRNA Control siRNA % mitochondrial fragmentation Milani et al. Cell Death Discovery (2019) 5:117 Page 5 of 11 Fig. 4 DRP-1 is not required for mitochondrial fission during BH3 mimetic-mediated apoptosis. H1299 cells were transfected with control, MCL-1, or BCL-X siRNAs, either alone or in combination with DRP-1 siRNA for 72 h, then exposed to Z-VAD.fmk (30 µM) for 0.5 h, followed by A- 1210477 (10 µM) and/or A-1331852 (100 nM) for 4 h and assessed for mitochondrial integrity by immunostaining with HSP70 antibody. The boxed regions in the images are enlarged to show mitochondrial structural changes in the indicated cells. Scale bar: 10 µm mediated by A-1210477 versus a combination of MCL-1 downregulated, A-1210477 resulted in mitochondrial and BCL-X inhibitors was distinct. Moreover, while structural changes that resembled matrix swelling (Fig. 4f, Supplementary Fig. S3), as previously described (Fig. 1d). S63845 failed to exhibit mitochondrial fission at low concentrations (100–1000 nM), higher concentrations In contrast, exposure to A-1331852 only resulted in (10 μM) of S63845 resulted in significant mitochondrial similar mitochondrial swelling when MCL-1 was also fission, which mimicked A-1210477-mediated mitochon- downregulated (Fig. 4g–I, Supplementary Fig. S3). These drial fragmentation (Fig. 3b). results suggested that mitochondrial fission, mediated by We previously reported that A-1210477-mediated MCL-1 inhibitors, appeared to exhibit a distinct mor- mitochondrial fission occurred in a DRP-1-dependent phology from that observed following the induction of manner . A similar dependence on DRP-1 was also apoptosis. This was more apparent following DRP-1 observed in cells exhibiting extensive mitochondrial fis- downregulation, which prevented A-1210477-mediated sion, following exposure to high concentrations of S63845 mitochondrial fission (Fig. 4m, n), but did not appear to (Fig. 3b). Thus both the MCL-1 inhibitors, A-1210477 and alter mitochondrial swelling observed during apoptosis S63845, induced mitochondrial fission, which was clearly induction (Fig. 4o, q, Supplementary Fig. S3). Taken dependent on DRP-1 (Fig. 3b). We wished to assess if together, these results exclude an involvement of DRP-1 such mitochondrial fission was a prerequisite for apop- in the early mitochondrial structural changes including tosis induction. Since H1299 cells depend on both BCL- mitochondrial swelling associated with the onset of X and MCL-1 for survival, we exposed cells to either A- apoptosis (Fig. 4). 1210477 or A-1331852 and simultaneously silenced the Consistent with the above hypothesis, electron micro- expression levels of either BCL-X or MCL-1 to facilitate graphs revealed marked structural alterations of the apoptosis. Although downregulation of BCL-XL or MCL- mitochondria in cells exposed to both A-121077 and A- 1 did not result in mitochondrial fission and maintained 1331852, characterised by breaks in the OMM (denoted the filamentous structure, exposure of the cells to A- by the yellow arrowheads), mitochondrial matrix swelling 1210477 resulted in significant mitochondrial fission, and a concomitant loss of cristae (Fig. 5a). Down- which resembled fragmented mitochondria (Fig. 4a–d, regulation of DRP-1 alone resulted in elongated mito- Supplementary Fig. S3). In the MCL-1-downregulated chondria, consistent with its known role in mitochondrial cells, A-1210477 still retained its ability to cause mito- fission (Fig. 5a). However, the mitochondria in the DRP-1- chondrial fragmentation (Fig. 4e), but when BCL-X was downregulated cells following exposure to BH3 mimetics Official journal of the Cell Death Differentiation Association Milani et al. Cell Death Discovery (2019) 5:117 Page 6 of 11 ab A-1210477+A-1331852 DMSO DRP-1 siRNA DRP-1 K38A Control siRNA DMSO A-1210477+A-1331852 Cyt. C *** *** cd e DMSO DMSO DMSO A-1210477+A-1331852 A-1210477+A-1331852 A-1210477+A-1331852 Cyt. C *** *** *** 100 *** 100 A-1210477+ *** A-1331852 - + - + *** 80 80 DRP-1 Cyt. C (cyto) 12 Cyt. C (mito) 12 40 40 OPA1 c 20 20 GAPDH 0 0 Fig. 5 DRP-1 regulates BH3 mimetic-induced cytochrome c release and apoptosis downstream of mitochondrial cristae remodelling. a Electron microscopy of H1299 cells, transfected with DRP-1 siRNA for 72 h in the presence or absence of the indicated BH3 mimetics for 2 h. Breaks in the outer mitochondrial membrane are indicated by the yellow arrowhead. Scale bars= 10 nm. b H1299 cells were transfected with DRP-1 siRNA or GFP-DRP-1 K38A plasmid for 72 h, exposed to Z-VAD.fmk (30 µM) for 0.5 h, followed by a combination of A-1331852 (100 nM) and A-1210477 (10 µM) for 4 h and the extent of cytochrome c released from mitochondria assessed by confocal microscopy. The boxed regions in the images are enlarged to show mitochondrial structural changes in the indicated cells. The extent of cytochrome c release was quantified by counting at least 100 cells from three independent experiments. c Same as b, but the extent of cytochrome c release as well as OPA1 processing and the silencing efficiency of DRP- 1 siRNA were analysed by western blotting. d H1299 cells were transfected with the indicated siRNAs for 72 h, treated as described in b and the extent of cytochrome c release assessed and quantified. The extent of cytochrome c release was quantified by counting at least 100 cells from three independent experiments. e Same as d but the cells were exposed to BH3 mimetics in the absence of Z-VAD.fmk and the extent of apoptosis assessed by PS externalisation from at least three independent experiments. All scale bars, unless indicated: 10 µm. Error bars= mean ± SEM. Statistical analysis was conducted by one-way ANOVA (***P ≤ 0.001) appeared visibly swollen with intact cristae and few if any Exposure of cells to a combination of A-1210477 and A- breaks in the OMM (Fig. 5a). Taken together, our data 1331852 resulted in an almost complete release of mito- suggested that mitochondrial fission observed following chondrial cytochrome c into the cytosol (Fig. 5b, c). This exposure to MCL-1 inhibitors was distinct from the was markedly inhibited in cells, following inactivation of structural perturbations (characterised by OMM breaks DRP-1 using siRNA or overexpression of the DRP-1 K38A and IMM swelling) observed as a result of apoptosis plasmid (Fig. 5b, c), thus placing DRP-1 upstream of induction. cytochrome c release. While cytochrome c was still retained in mitochondria following DRP-1 down- DRP-1 is critical for the release of cytochrome c from regulation, mitochondria in these cells appeared swollen mitochondria during apoptosis (Fig. 5b), consistent with those observed in the electron Permeabilisation of the OMM, otherwise known as micrographs (Fig. 5a). As cytochrome c release occurs as a MOMP, occurs as a consequence of BAX and/or BAK consequence of mitochondrial cristae remodelling , oligomerization and is generally accompanied by the exposure to BH3 mimetics not only resulted in the release release of mitochondrial cytochrome c into the cytosol. of mitochondrial cytochrome c but also caused a loss of Official journal of the Cell Death Differentiation Association Control si DRP-1 si Control siRNA DRP-1 siRNA DRP-1 K38A Control si DRP-1 si OPA1 si DRP-1 + OPA1 si Control si DRP-1 si OPA1 si DRP-1 + OPA1 si DRP-1 siRNA Control siRNA DRP-1 + OPA1 siRNA OPA1 siRNA A-1210477 + A-1331852 DMSO % Cyt.C release % PS externalisation % Cyt.C release Milani et al. Cell Death Discovery (2019) 5:117 Page 7 of 11 the high molecular weight isoforms of OPA1, character- DRP-1 is critical for BAK activation during BH3 mimetic- istic of mitochondrial cristae remodelling (Fig. 5c). While mediated apoptosis downregulation of DRP-1 markedly diminished BH3 Our results indicated that BH3 mimetics could induce mimetic-mediated release of cytochrome c, it did not structural perturbations in the mitochondria, char- prevent BH3 mimetic-mediated loss of OPA1 (Fig. 5c), acterised by OPA1 proteolysis, cristae remodelling and the thus placing DRP-1 upstream of cytochrome c release but accompanying redistribution of cytochrome c from cristae downstream of mitochondrial cristae remodelling. This to mitochondrial inner membrane space, all irrespective of was further confirmed following exposure of DRP-1 and/ the presence or absence of DRP-1. Since the role of DRP-1 or OPA1-downregulated cells to BH3 mimetics. While was placed upstream of cytochrome c release, we wished downregulation of OPA1 resulted in significant mito- to assess whether DRP-1 impacted on any upstream events chondrial fission, as well as a near-complete release of during BH3 mimetic-mediated apoptosis. The primary cytochrome c following BH3 mimetics, a simultaneous function of BH3 mimetics is to disrupt the protein–protein downregulation of DRP-1 diminished these effects (Fig. interactions between the anti-apoptotic (BCL-X and 5d). Similarly downregulation of DRP-1 prevented BH3 MCL-1, in this instance) and pro-apoptotic members of mimetic-induced apoptosis, even in the absence of OPA1 the BCL-2 family. The released pro-apoptotic proteins (Fig. 5e), thus placing DRP-1 downstream of OPA1 pro- could then activate the effector proteins (BAK, in H1299 teolysis but upstream of cytochrome c release in BH3 as these cells lack BAX) to oligomerise on mitochondrial mimetic-mediated apoptosis. membranes to subsequently release cytochrome c (Fig. 6a). Fig. 6 DRP-1 regulates BH3 mimetic-induced activation of BAK. a Scheme demonstrating the primary mechanism of action of BH3 mimetics and the downstream events that culminate in apoptosis. b Immunoprecipitation of BCL-X and MCL-1 was carried out in H1299 cells, transfected with control or DRP-1 siRNA, followed by exposure to Z-VAD.fmk (30 µM) for 0.5 h and a combination of A-1331852 (100 nM) and A-1210477 (10 µM) for 4 h, and the eluted complexes were immunoblotted for the indicated proteins. c H1299 cells were treated as b and the extent of BAK activation was assessed by flow cytometry from at least three independent experiments. Error bars = mean ± SEM. Statistical analysis was conducted by one-way ANOVA (***P ≤ 0.001). d Western blots of different molecular weight fractions from size exclusion chromatography showing BAK oligomerisation in H1299 cells upon exposure to A-1331852 (100 nM) and A-1210477 (10 µM) for 2 h. e Immunoprecipitation of active BAK in H1299 cells treated as b, and the eluted complexes were immunoblotted for the indicated proteins. f Representative images of cells showing BAK activation and DRP-1 distribution in H1299 cells treated as b. Scale bar: 10 µm Official journal of the Cell Death Differentiation Association Milani et al. Cell Death Discovery (2019) 5:117 Page 8 of 11 Immunoprecipitation of BCL-X and MCL-1 to identify ascertain whether such fission was a prerequisite for their associated pro-apoptotic proteins revealed that in apoptosis. This difficulty was partly because DRP-1 H1299 cells, BIM and BAK were bound to both BCL-X appeared to play important but distinct roles both in A- and MCL-1, whereas NOXA and BAD exclusively bound 1210477-mediated mitochondrial fission and BH3 to MCL-1 and BCL-X , respectively (Fig. 6b). Exposure of mimetic-mediated apoptosis . Moreover, DRP-1 also these cells to a combination of A-1331852 and A-1210477 interacted with MCL-1 and BCL-X , thus coupling 23,27,36 resulted in displacement of most of these pro-apoptotic mitochondrial fission and apoptosis . However, with proteins from their corresponding ant-apoptotic partners the development of more potent inhibitors, such as (Fig. 6b). Importantly, none of these interactions/dis- S63845, we have demonstrated that mitochondrial fission placements were altered in cells following DRP-1 down- does not occur at concentrations sufficient to inhibit regulation, thus suggesting that DRP-1 played no role in MCL-1 (Fig. 3). Furthermore, while mitochondrial fission the early events of BH3 mimetic-mediated apoptosis. Since induced by A-1210477 and high concentrations of S63845 BAK and other BH3-only proteins were released following was mediated by DRP-1, mitochondrial swelling that BH3 mimetics, we next wished to assess if BAK activation occurred at the onset of apoptosis induction was largely was altered in the absence of DRP-1. Downregulation of independent of DRP-1 (Figs. 3 and 4), thus differentiating DRP-1 resulted in a significant decrease in BH3 mimetic- the distinct types of mitochondrial fission. mediated activation of BAK (Fig. 6c), suggesting that DRP- Our data in the HCT-116 WT and BAX/BAK DKO 1 was critical in the activation of BAK during BH3 cells convincingly demonstrate that BH3 mimetic- mimetic-mediated apoptosis. Although the requirement of mediated OMM breaks and swelling of matrix compart- DRP-1 for BAK activation could be demonstrated, no ment are essential for BAX/BAK to facilitate cytochrome binding of DRP-1 to the oligomerised/active BAK was c release (Fig. 2). The inability of HCT-116 OctaKO cells observed in these cells (Fig. 6d–f), thus suggesting the to prevent BH3 mimetic-mediated mitochondrial changes involvement of other protein(s) in BAK activation imme- further supports our findings that BAX and BAK but not diately preceding cytochrome c release. Taken together, the known BH3-only members are critical for BH3 33,34 our data confirm that DRP-1 plays a critical role at the mimetic-mediated apoptosis . How BAX and BAK level of BAK activation, facilitating OMM breaks, cyto- localise to the sites of OMM breaks to facilitate cyto- chrome c release and apoptosis. chrome c release is not entirely known. The involvement of DRP-1, Dynamin-2, and even membranes of the endoplasmic reticulum in these events have been pre- Discussion 37–42 BH3 mimetics, in particular ABT-737 and ABT-199, viously proposed . Downregulation of DRP-1 or its induce a novel paradigm of cell death, characterised by receptors, MID49 and MID51, have been shown to excessive swelling of mitochondrial matrix and dis- antagonise cytochrome c release and apoptosis in continuities in the OMM in BCL-2-dependent chronic response to a wide variety of apoptotic stimuli . DRP-1 19,31 lymphocytic leukaemia cells . BH3 mimetics targeting functions downstream of OPA1-mediated cristae remo- BCL-X and MCL-1 also induce similar mitochondrial delling (Fig. 5), to activate BAK (Fig. 6), which in turn ultrastructural changes in cells that exclusively depend on precedes BAK oligomerisation and membrane insertion BCL-X and MCL-1, respectively (Fig. 1) . However, cells for the execution of MOMP and apoptosis. However, exposed to the MCL-1 inhibitor, A-1210477, exhibit mitochondrial cristae remodelling requires the presence marked mitochondrial changes, in particular mitochon- of BAX and BAK (Fig. 2) . Thus DRP-1 could function drial fission, irrespective of their dependencies on a spe- either downstream or independent of OPA1 proteolysis to cific BCL-2 family member for survival (Fig. 3). This is in activate BAK and ensuing apoptosis. Taken together, our agreement with our previous findings . Mitochondrial data suggest that BH3 mimetics most likely activate BAX/ fission mediated by A-1210477 alone did not result in BAK independently of the eight known BH3-only mem- apoptosis in these cell lines, even after prolonged expo- bers, which further results in OPA1-mediated cristae sure . This was most probably because most cell lines remodelling to redistribute cytochrome c within the derived from solid tumours depend on both BCL-X and mitochondria, thus priming the mitochondria to undergo MCL-1 for survival, and inhibition of MCL-1 alone was MOMP, upon sensing the stress signal. DRP-1 plays a not sufficient to result in apoptosis. This was further critical role at this stage to activate BAK and/or BAX to supported by our observation that inhibition of MCL-1 insert these effector proteins on mitochondrial mem- using A-1210477 while resulting in extensive mitochon- branes. This along with the constriction of the primed drial fission did not induce OMM breaks and cell death, mitochondria by DRP-1 constitute the so-called stress unless the activity of BCL-X was also neutralised. signals that cause OMM breaks, efficiently releasing the Although A-1210477-mediated mitochondrial fission redistributed cytochrome c into the cytosol and initiating did not necessarily result in apoptosis, it was difficult to apoptosis. Official journal of the Cell Death Differentiation Association Milani et al. Cell Death Discovery (2019) 5:117 Page 9 of 11 Materials and methods metal staining, which consisted of two consecutive Cell culture osmium tetroxide steps (2% (w/v) OsO4 in ddh O), fol- H1299 (purchased from ATCC), K562 (provided by lowed by 1% (w/v) aqueous uranyl acetate. To prevent Prof. R. Clark, University of Liverpool) and MAVER-1 precipitation artefacts, the cells were washed copiously cells (provided by Dr. J. Slupsky, University of Liverpool) with ddH O between each staining step. All fixation and were cultured in RPMI 1640 medium (Life Technologies). staining steps were performed in a Pelco Biowave®Pro H929 cells (purchased from DMSZ, Braunshweig, (Ted Pella Inc., Redding, California, USA) at 100w 20Hg, Germany) were cultured in RPMI 1640 medium supple- for 3 min and 1 min, respectively. Dehydration was in a mented with 0.05 mM β-mercaptoethanol (BME). Colon graded ethanol series before filtration and embedding in cancer cell lines HCT-116 (wild-type and DKO) (from R. medium premix resin (TAAB, Reading, UK). Seventy to J. Youle, National Institute of Health, USA) and HCT- 74 nm serial sections were cut using a UC6 ultra micro- 116-OctaKO were cultured in McCoy’s 5A Modified tome (Leica Microsystems, Wetzlar, Germany) and col- media. All culture media were supplemented with 10% lected on Formvar (0.25% (w/v) in chloroform (TAAB, FBS (Life Technologies) and maintained at 37 °C in a Reading, UK) coated Gilder 200 mesh copper grids humidified atmosphere of 5% CO . All cell lines used in (GG017/C; TAAB, Reading, UK). Images were acquired this study were subjected to short tandem repeat (STR) on a 120 kV Tecnai G2 Spirit BioTWIN (FEI, Hillsboro, profiling to ensure quality and integrity. Oregon, USA) using a MegaView III camera and analySIS software (Olympus, Germany). For immunocytochem- Reagents istry, cells grown on coverslips were fixed with 4% (w/v) ABT-199, A-1210477 and Z-VAD.FMK from Selleck paraformaldehyde, permeabilised with 0.5% (v/v) Triton (Houston, TX, USA), S63845 from Active Biochem X-100 in PBS, followed by incubations with primary (Kowloon, Hong Kong) and A-1331852 from AbbVie Inc. antibodies (diluted 1:250 in 3% BSA in PBS), the appro- (North Chicago, IL, USA) were used. Antibodies against priate fluorophore-conjugated secondary antibodies HSP70 (cat#ab2799) from Abcam (Cambridge, UK), (diluted 1:1000 in 3% BSA in PBS), mounted on glass OPA1 (cat#612607), cytochrome c (cat#556432) and slides and imaged using a 3i Marianas spinning disk DRP-1 (cat#611113) from BD Biosciences (San Jose, CA, confocal microscope, fitted with a Plan-Apochromat ×63/ USA); BCL-X (cat#2762), BIM (cat#2933) and BAD 1.4 NA oil objective, M27 and a Hamamatsu ORCA- (cat#9292) from Cell Signalling Technology (MA, USA); Flash4.0 v2 sCMOS Camera (all from Intelligent Imaging BAK (AB-1) (cat#AM-03) and NOXA (cat#OP180) from Innovations, GmbH, Germany). Millipore (Watford, UK) and MCL-1 (cat#sc-819), BAK (cat#sc-832) and GAPDH (cat#sc-25778) from Santa Cruz Cytochrome c release assay Biotechnologies (Santa Cruz, CA, USA) were used. All Approximately 10 cells were washed in cold PBS and other reagents were obtained from Sigma Aldrich (St. resuspended in mitochondrial isolation buffer (250 mM Louis, MO, USA). sucrose, 20 mM HEPES, pH 7.4, 5 mM MgCl and 10 mM KCl) containing 0.01% digitonin. Cells were left on ice Overexpression and genetic silencing for 5 min followed by centrifugation at 13000 g for 3 min For transient overexpression studies, cells were trans- at 4 °C. Subsequently, the supernatant (cytosolic fraction) fected with GFP-DRP1 K38A plasmid (provided by Dr. E. and pellet (mitochondrial fraction) were processed for Bampton, University of Leicester, UK), using TransIT-LT- western blotting. 1 transfection reagent (Mirus Bio LLC, Madison, WI, USA), according to the manufacturer’s protocol. For RNA Size exclusion chromatography, immunoprecipitation and interference, cells were transfected with 10 nM of siRNAs western blotting against DRP-1 (s104274235), MCL-1 (s8585 or Size exclusion chromatography and immunoprecipi- SI02781205) or BCL-X siRNA (s1920) purchased from tation experiments were carried out as previously 16,27 Qiagen Ltd (Manchester, UK) or ThermoFisher Scientific described . Western blotting was carried out (Waltham, MA, USA). Cells were transfected using 0.33% according to standard protocols. Briefly, 50 μgoftotal (v/v) Interferin reagent (Polyplus Transfection Inc., NY) protein lysate was subjected to SDS-PAGE electro- to culture media, according to the manufacturer’s proto- phoresis. Subsequently proteins were transferred to col and processed 72 h after transfection. nitrocellulose membrane, probed with appropriate pri- mary antibodies (diluted 1:1000 in Tris-buffered saline Microscopy with 0.1% Tween-20), species-specific secondary anti- For electron microscopy, cells were fixed in 2.5% (w/v) bodies (diluted 1:2000 in Tris-buffered saline with 0.1% glutaraldehyde and 2 mM calcium chloride in 0.1 M Tween-20) and protein bands visualised with ECL cacodylate buffer (pH 7.4). This was followed by heavy reagents (GE Healthcare). Official journal of the Cell Death Differentiation Association Milani et al. Cell Death Discovery (2019) 5:117 Page 10 of 11 Received: 5 June 2019 Revised: 17 June 2019 Accepted: 23 June 2019 Flow cytometry The extent of apoptosis in cells following different treatments was quantified by using an Attune NxT flow cytometer (ThermoFisher Scientific, Paisley, UK) follow- References ing staining of the cells with AnnexinV-FITC and propi- 1. 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DRP-1 functions independently of mitochondrial structural perturbations to facilitate BH3 mimetic-mediated apoptosis

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Copyright © 2019 by The Author(s)
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Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Stem Cells; Apoptosis; Cell Cycle Analysis
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Abstract

Maintenance of mitochondrial integrity is critical for normal cellular homoeostasis. Most cells respond to stress stimuli and undergo apoptosis by perturbing mitochondrial structure and function to release proteins, such as cytochrome c, which are essential for the execution of the intrinsic apoptotic cascade. Cancer cells evade these events by overexpressing the anti-apoptotic BCL-2 family of proteins on mitochondrial membranes. Inhibitors of the anti- apoptotic BCL-2 family proteins, also known as BH3 mimetics, antagonise the pro-survival functions of these proteins and result in rapid apoptosis. Although the precise mechanism by which BH3 mimetics induce apoptosis has been well characterised, not much is known in terms of the structural changes that occur in mitochondria during apoptosis. Using a panel of highly selective BH3 mimetics and a wide range of cell lines, we demonstrate that BH3 mimetics induce extensive mitochondrial fission, accompanied by swelling of the mitochondrial matrix and rupture of the outer mitochondrial membrane. These changes occur in a BAX/ BAK-dependent manner. Although a major mitochondrial fission GTPase, DRP-1, has been implicated in mitochondrial apoptosis, our data demonstrate that DRP-1 might function independently/downstream of BH3 mimetic-mediated mitochondrial fission to facilitate the release of cytochrome c and apoptosis. Moreover, downregulation of DRP-1 prevented cytochrome c release and apoptosis even when OPA1, a protein mediating mitochondrial fusion, was silenced. Although BH3 mimetic-mediated displacement of BAK and other BH3-only proteins from BCL-X and MCL-1 was unaffected by DRP-1 downregulation, it prevented BAK activation significantly, thus placing DRP-1 as one of the most critical players, along with BAX and BAK, that governs BH3 mimetic-mediated cytochrome c release and apoptosis. Introduction mitochondrial membrane (IMM) and formation of the Most chemotherapeutic agents kill cancer cells by apoptosome that activates the initiator and effector cas- executing the intrinsic apoptotic pathway, which is char- pases. MOMP is regulated by the BCL-2 family, whereby acterised by mitochondrial outer membrane permeabili- BAX and BAK, undergo specific conformational changes zation (MOMP), release of cytochrome c from the inner to form oligomeric pores that insert into the outer mitochondrial membrane (OMM) to release cytochrome 1,2 c . Activation of BAX and BAK is achieved by several Correspondence: Shankar Varadarajan (svar@liv.ac.uk) pro-apoptotic BH3-only members, which are generally Department of Molecular and Clinical Cancer Medicine, Institute of rendered ineffective by sequestration with specific anti- Translational Medicine, University of Liverpool, Liverpool, Ashton Street, apoptotic BCL-2 family of proteins, such as BCL-2, BCL- Liverpool L69 3GE, UK 2 3,4 Department of Cellular and Molecular Physiology, Institute of Translational X and MCL-1 . These anti-apoptotic proteins are Medicine, University of Liverpool, Liverpool, Ashton Street, Liverpool L69 3GE, highly expressed in many cancers and inhibitors known as UK BH3 mimetics have been designed to target them in order Full list of author information is available at the end of the article. Edited by I. Amelio © 2019 The Author(s). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Milani et al. Cell Death Discovery (2019) 5:117 Page 2 of 11 to displace the BH3-only proteins, activate BAX and BAK, Results thereby inducing MOMP and apoptosis of cancer cells . BH3 mimetics induce marked mitochondrial structural ABT-737, and its orally available analogue, ABT-263 changes (Navitoclax) were the first bona fide BH3 mimetics Previously, we have reported that BH3 mimetics induce 6,7 developed to target BCL-2, BCL-X and BCL-w . Sub- a novel paradigm of apoptosis characterised by marked sequently, BH3 mimetics that specifically target BCL-2 ultrastructural changes in the mitochondria, involving the (ABT-199 (Venetoclax), S55746), BCL-X (A-1331852) loss of mitochondrial cristae and the appearance of breaks and MCL-1 (A-1210477, S63845, AMG 176 and in the OMM, resulting from mitochondrial matrix swel- 8–14 19,20,31 AZD5991) have been synthesised . These inhibitors, as ling . In cell lines that depend for survival almost single agents, have demonstrated much promise in treat- exclusively on BCL-2 (MAVER-1), BCL-X -(K562) and ing a wide variety of haematological malignancies, and MCL-1 (H929) , exposure to the relevant BH3 mimetics, have had limited success in combination with conven- such as ABT-199, A-1331852 and A-1210477, respec- 8,12–17 tional chemotherapy against several solid tumours . tively, resulted in similar mitochondrial matrix swelling BH3 mimetics induce apoptosis primarily by targeting and rupture of the OMM (Fig. 1a–c). Such mitochondrial protein–protein interactions between the anti- and pro- changes were also evident in H1299 cells following apoptotic BCL-2 family members . Subsequently, BH3 exposure to a combination of A-1331852 and A-1210477, mimetics have been shown to induce significant structural as these cells depend on both BCL-X and MCL-1 for changes in the mitochondria, ranging from mitochondrial survival (Fig. 1d). These mitochondrial ultrastructural matrix swelling to discontinuities in the OMM, upstream changes were independent of effector caspases, as they 19,20 of caspase activation . Furthermore, BAX and BAK were observed in cells pre-treated with Z-VAD.fmk, a localise to the breakpoints in OMM and may facilitate broad-spectrum caspase inhibitor (Fig. 1). Exposure of the cytochrome c release at such breakpoints . Although different cells to their appropriate BH3 mimetic resulted BCL-2 family members have been implicated in regulating in mitochondrial membrane depolarisation, loss of cyto- 21–24 mitochondrial membrane dynamics and functions , chrome c and induction of apoptosis, as assessed by putative inhibitors of MCL-1 have often resulted in phosphatidylserine externalisation (Supplementary Fig. 25–27 extensive mitochondrial fission in various cell lines . S1). Exposure of the cells to Z-VAD.fmk almost com- The regulation of this fission and its relationship to BH3 pletely inhibited BH3 mimetic-mediated apoptosis, mimetic-mediated apoptosis remains to be determined. assessed by PS externalisation, whereas little if any inhi- Mitochondrial structure is maintained through an bition of cytochrome c release was observed (Supple- intricate balance between the activities of several fusion mentary Fig. S1). Taken together these results suggested and fission proteins, which belong to a conserved family that the mitochondrial structural changes occurred of GTPases that reside in the OMM or IMM. Mitofusins 1 upstream of effector caspase activation and accompanied and 2 (MFN1/ 2) and optic atrophy 1 (OPA1) are essential cytochrome c release, as well as a loss of mitochondrial for mitochondrial fusion, whereas dynamin related pro- membrane potential. tein 1 (DRP-1) is essential for mitochondrial fission . Defects in mitochondrial fusion and fission have been BH3 mimetic-mediated mitochondrial perturbations occur implicated in a range of pathophysiological conditions in a BAX/BAK-dependent manner including poor brain development, optic atrophy, cardi- To assess whether BAX and BAK play crucial roles in 29,30 omyopathy and neurodegenerative diseases . Mount- BH3 mimetic-mediated ultrastructural changes in mito- ing evidence now suggests the involvement of several chondria, we exposed HCT-116 WT and BAX/BAK members of BCL-2 family members, particularly MCL-1, double knock-out (DKO) cells to a combination of A- in the regulation of mitochondrial structure and func- 1331852 and A-1210477, as HCT-116 cells also depend 22–25,29 33 tion . However, the mechanism by which MCL-1 on both BCL-X and MCL-1 for survival . Exposure of regulates mitochondrial membrane dynamics and the the HCT-116 WT cells to the BH3 mimetics resulted in potential cross-talk with its conventional role in antag- significant mitochondrial matrix swelling accompanied by onising apoptosis remain to be characterised. a loss of mitochondrial cristae, although rupture of the In this study, we use a panel of highly selective BH3 OMM was not readily apparent (Fig. 2). However, all mimetics together with cell lines that depend on specific these mitochondrial changes were clearly prevented in the BCL-2 family members for survival to demonstrate that HCT-116 BAX/BAK DKO cells, demonstrating a BH3 mimetics induce significant ultrastructural mito- requirement for BAX and/or BAK for the perturbation of chondrial changes upstream of caspase activation. DRP-1 the mitochondria. Since BH3-only members are generally plays a role downstream of these changes but upstream of required to activate BAX and BAK, we wished to assess MOMP to facilitate cytochrome c release and apoptosis, whether BH3 mimetics could induce mitochondrial following exposure to BH3 mimetics. structural perturbations in the absence of all known pro- Official journal of the Cell Death Differentiation Association Milani et al. Cell Death Discovery (2019) 5:117 Page 3 of 11 Fig. 1 BH3 mimetics induce marked ultrastructural changes in mitochondria of different cells. a MAVER-1, b K562, c H929 and d H1299 cells were exposed to Z-VAD.fmk (30 µM) for 0.5 h, followed by ABT-199 (100 nM), A-1331852 (100 nM), A-1210477 (10 µM), or a combination of A-1331852 (100 nM) and A-1210477 (10 µM), respectively, for 4 h and assessed for mitochondrial structural changes by electron microscopy. Yellow arrowheads indicate regions of breaks at the outer mitochondrial membrane. Scale bars: 500 nm HCT-116 OctaKO HCT-116 WT HCT-116 BAX/BAK DKO Fig. 2 BH3 mimetics disrupt mitochondria in a BAX- and BAK-dependent but BH3-independent manner. HCT-116 WT, DKO (BAX/BAK deficient) and OctaKO cells were exposed to Z-VAD.fmk (30 µM) for 0.5 h, followed by a combination of A-1331852 (100 nM) and A-1210477 (10 µM) for 4 h and assessed for mitochondrial structural changes by electron microscopy. Scale bars: 500 nm Official journal of the Cell Death Differentiation Association A-1210477+A-1331852 DMSO Milani et al. Cell Death Discovery (2019) 5:117 Page 4 of 11 DMSO A-1331852 A-1210477 A-1331852+A-1210477 S63845 A-1331852+S63845 DMSO A-1210477 S63845 (1 uM) S63845 (10 uM) Control siRNA DRP-1 siRNA *** *** Fig. 3 Mitochondrial fission mediated by A-1210477 and S63845 occurs in a DRP-1-dependent manner. a H1299 cells were exposed to Z- VAD.fmk (30 µM) for 0.5 h, followed by either A-1331852 (100 nM), A-1210477 (10 µM), S63845 (100 nM), or a combination of the different inhibitors for 4 h and assessed for mitochondrial integrity by immunostaining with HSP70 antibody. b H1299 cells were transfected with control or DRP-1 siRNA for 72 h and exposed to A-1210477 (10 µM) or S63845 (1 and 10 µM) for 4 h and assessed for mitochondrial integrity. The extent of mitochondrial fragmentation was quantified by analysing ~100 cells for each condition in three independent experiments. Scale bar: 10 µm. Error bars = mean ± SEM. Statistical analysis was conducted by one-way ANOVA (***P ≤ 0.001) apoptotic BH3-only members. For this, we used HCT-116 suggested that this could be a prerequisite for the ensuing 25–27 OctaKO cells, which lack the BH3-only members namely, apoptosis in MCL-1-dependent cell lines . In support BIM, BID, PUMA, BAD, BIK, HRK, BMF and NOXA . of this suggestion, exposure of A-1210477 but not A- Exposure of these cells to a combination of A-1331852 1331852 resulted in extensive mitochondrial fission that and A-1210477 resulted in mitochondrial structural resembled mitochondrial fragmentation in H1299 cells changes, characteristic of significant cristae remodelling (Fig. 3a, Supplementary Fig. S2). The ability of A-1210477 (Fig. 2). However, the swelling of mitochondrial matrix to induce mitochondrial fission was also clearly evident and the accompanying loss of cristae observed in the when used in combination with A-1331852 to induce HCT-116 WT cells following BH3 mimetics were not apoptosis in these cells (Fig. 3a, Supplementary Fig. S2). apparent in HCT-116 OctaKO cells (Fig. 2). This is con- However, mitochondria in this instance appeared swollen, sistent with earlier findings demonstrating that the pro- potentially indicating swollen matrix and loss of cristae apoptotic BH3-only members are dispensable for BH3 that were previously observed at the level of electron mimetic-mediated apoptosis . Taken together, our data microscopy (compare Figs. 1d and 3a). In marked con- demonstrated that the activation of BAX and/or BAK, trast, S63845 at a concentration (100 nM) sufficient to either in a BH3-dependent or independent manner, is induce apoptosis in a MCL-1-dependent manner failed essential for the ultrastructural changes observed in the to demonstrate mitochondrial fission (Fig. 3a, Supple- mitochondria, following exposure to BH3 mimetics. mentary Fig. S2). However, S63845 (100 nM) when used in conjunction with A-1331852 resulted in mitochondrial DRP-1 is not required for the mitochondrial structural structural changes that resembled the swollen mito- changes that occur during the onset of apoptosis chondria observed following a combination of A-1210477 We previously reported that putative inhibitors of and A-1331852 (Fig. 3a, Supplementary Fig. S2). Taken MCL-1 induced extensive mitochondrial fission and together, our results suggested that mitochondrial fission Official journal of the Cell Death Differentiation Association DMSO A-1210477 S63845 (1 uM) S63845 (10 uM) DRP-1 siRNA Control siRNA % mitochondrial fragmentation Milani et al. Cell Death Discovery (2019) 5:117 Page 5 of 11 Fig. 4 DRP-1 is not required for mitochondrial fission during BH3 mimetic-mediated apoptosis. H1299 cells were transfected with control, MCL-1, or BCL-X siRNAs, either alone or in combination with DRP-1 siRNA for 72 h, then exposed to Z-VAD.fmk (30 µM) for 0.5 h, followed by A- 1210477 (10 µM) and/or A-1331852 (100 nM) for 4 h and assessed for mitochondrial integrity by immunostaining with HSP70 antibody. The boxed regions in the images are enlarged to show mitochondrial structural changes in the indicated cells. Scale bar: 10 µm mediated by A-1210477 versus a combination of MCL-1 downregulated, A-1210477 resulted in mitochondrial and BCL-X inhibitors was distinct. Moreover, while structural changes that resembled matrix swelling (Fig. 4f, Supplementary Fig. S3), as previously described (Fig. 1d). S63845 failed to exhibit mitochondrial fission at low concentrations (100–1000 nM), higher concentrations In contrast, exposure to A-1331852 only resulted in (10 μM) of S63845 resulted in significant mitochondrial similar mitochondrial swelling when MCL-1 was also fission, which mimicked A-1210477-mediated mitochon- downregulated (Fig. 4g–I, Supplementary Fig. S3). These drial fragmentation (Fig. 3b). results suggested that mitochondrial fission, mediated by We previously reported that A-1210477-mediated MCL-1 inhibitors, appeared to exhibit a distinct mor- mitochondrial fission occurred in a DRP-1-dependent phology from that observed following the induction of manner . A similar dependence on DRP-1 was also apoptosis. This was more apparent following DRP-1 observed in cells exhibiting extensive mitochondrial fis- downregulation, which prevented A-1210477-mediated sion, following exposure to high concentrations of S63845 mitochondrial fission (Fig. 4m, n), but did not appear to (Fig. 3b). Thus both the MCL-1 inhibitors, A-1210477 and alter mitochondrial swelling observed during apoptosis S63845, induced mitochondrial fission, which was clearly induction (Fig. 4o, q, Supplementary Fig. S3). Taken dependent on DRP-1 (Fig. 3b). We wished to assess if together, these results exclude an involvement of DRP-1 such mitochondrial fission was a prerequisite for apop- in the early mitochondrial structural changes including tosis induction. Since H1299 cells depend on both BCL- mitochondrial swelling associated with the onset of X and MCL-1 for survival, we exposed cells to either A- apoptosis (Fig. 4). 1210477 or A-1331852 and simultaneously silenced the Consistent with the above hypothesis, electron micro- expression levels of either BCL-X or MCL-1 to facilitate graphs revealed marked structural alterations of the apoptosis. Although downregulation of BCL-XL or MCL- mitochondria in cells exposed to both A-121077 and A- 1 did not result in mitochondrial fission and maintained 1331852, characterised by breaks in the OMM (denoted the filamentous structure, exposure of the cells to A- by the yellow arrowheads), mitochondrial matrix swelling 1210477 resulted in significant mitochondrial fission, and a concomitant loss of cristae (Fig. 5a). Down- which resembled fragmented mitochondria (Fig. 4a–d, regulation of DRP-1 alone resulted in elongated mito- Supplementary Fig. S3). In the MCL-1-downregulated chondria, consistent with its known role in mitochondrial cells, A-1210477 still retained its ability to cause mito- fission (Fig. 5a). However, the mitochondria in the DRP-1- chondrial fragmentation (Fig. 4e), but when BCL-X was downregulated cells following exposure to BH3 mimetics Official journal of the Cell Death Differentiation Association Milani et al. Cell Death Discovery (2019) 5:117 Page 6 of 11 ab A-1210477+A-1331852 DMSO DRP-1 siRNA DRP-1 K38A Control siRNA DMSO A-1210477+A-1331852 Cyt. C *** *** cd e DMSO DMSO DMSO A-1210477+A-1331852 A-1210477+A-1331852 A-1210477+A-1331852 Cyt. C *** *** *** 100 *** 100 A-1210477+ *** A-1331852 - + - + *** 80 80 DRP-1 Cyt. C (cyto) 12 Cyt. C (mito) 12 40 40 OPA1 c 20 20 GAPDH 0 0 Fig. 5 DRP-1 regulates BH3 mimetic-induced cytochrome c release and apoptosis downstream of mitochondrial cristae remodelling. a Electron microscopy of H1299 cells, transfected with DRP-1 siRNA for 72 h in the presence or absence of the indicated BH3 mimetics for 2 h. Breaks in the outer mitochondrial membrane are indicated by the yellow arrowhead. Scale bars= 10 nm. b H1299 cells were transfected with DRP-1 siRNA or GFP-DRP-1 K38A plasmid for 72 h, exposed to Z-VAD.fmk (30 µM) for 0.5 h, followed by a combination of A-1331852 (100 nM) and A-1210477 (10 µM) for 4 h and the extent of cytochrome c released from mitochondria assessed by confocal microscopy. The boxed regions in the images are enlarged to show mitochondrial structural changes in the indicated cells. The extent of cytochrome c release was quantified by counting at least 100 cells from three independent experiments. c Same as b, but the extent of cytochrome c release as well as OPA1 processing and the silencing efficiency of DRP- 1 siRNA were analysed by western blotting. d H1299 cells were transfected with the indicated siRNAs for 72 h, treated as described in b and the extent of cytochrome c release assessed and quantified. The extent of cytochrome c release was quantified by counting at least 100 cells from three independent experiments. e Same as d but the cells were exposed to BH3 mimetics in the absence of Z-VAD.fmk and the extent of apoptosis assessed by PS externalisation from at least three independent experiments. All scale bars, unless indicated: 10 µm. Error bars= mean ± SEM. Statistical analysis was conducted by one-way ANOVA (***P ≤ 0.001) appeared visibly swollen with intact cristae and few if any Exposure of cells to a combination of A-1210477 and A- breaks in the OMM (Fig. 5a). Taken together, our data 1331852 resulted in an almost complete release of mito- suggested that mitochondrial fission observed following chondrial cytochrome c into the cytosol (Fig. 5b, c). This exposure to MCL-1 inhibitors was distinct from the was markedly inhibited in cells, following inactivation of structural perturbations (characterised by OMM breaks DRP-1 using siRNA or overexpression of the DRP-1 K38A and IMM swelling) observed as a result of apoptosis plasmid (Fig. 5b, c), thus placing DRP-1 upstream of induction. cytochrome c release. While cytochrome c was still retained in mitochondria following DRP-1 down- DRP-1 is critical for the release of cytochrome c from regulation, mitochondria in these cells appeared swollen mitochondria during apoptosis (Fig. 5b), consistent with those observed in the electron Permeabilisation of the OMM, otherwise known as micrographs (Fig. 5a). As cytochrome c release occurs as a MOMP, occurs as a consequence of BAX and/or BAK consequence of mitochondrial cristae remodelling , oligomerization and is generally accompanied by the exposure to BH3 mimetics not only resulted in the release release of mitochondrial cytochrome c into the cytosol. of mitochondrial cytochrome c but also caused a loss of Official journal of the Cell Death Differentiation Association Control si DRP-1 si Control siRNA DRP-1 siRNA DRP-1 K38A Control si DRP-1 si OPA1 si DRP-1 + OPA1 si Control si DRP-1 si OPA1 si DRP-1 + OPA1 si DRP-1 siRNA Control siRNA DRP-1 + OPA1 siRNA OPA1 siRNA A-1210477 + A-1331852 DMSO % Cyt.C release % PS externalisation % Cyt.C release Milani et al. Cell Death Discovery (2019) 5:117 Page 7 of 11 the high molecular weight isoforms of OPA1, character- DRP-1 is critical for BAK activation during BH3 mimetic- istic of mitochondrial cristae remodelling (Fig. 5c). While mediated apoptosis downregulation of DRP-1 markedly diminished BH3 Our results indicated that BH3 mimetics could induce mimetic-mediated release of cytochrome c, it did not structural perturbations in the mitochondria, char- prevent BH3 mimetic-mediated loss of OPA1 (Fig. 5c), acterised by OPA1 proteolysis, cristae remodelling and the thus placing DRP-1 upstream of cytochrome c release but accompanying redistribution of cytochrome c from cristae downstream of mitochondrial cristae remodelling. This to mitochondrial inner membrane space, all irrespective of was further confirmed following exposure of DRP-1 and/ the presence or absence of DRP-1. Since the role of DRP-1 or OPA1-downregulated cells to BH3 mimetics. While was placed upstream of cytochrome c release, we wished downregulation of OPA1 resulted in significant mito- to assess whether DRP-1 impacted on any upstream events chondrial fission, as well as a near-complete release of during BH3 mimetic-mediated apoptosis. The primary cytochrome c following BH3 mimetics, a simultaneous function of BH3 mimetics is to disrupt the protein–protein downregulation of DRP-1 diminished these effects (Fig. interactions between the anti-apoptotic (BCL-X and 5d). Similarly downregulation of DRP-1 prevented BH3 MCL-1, in this instance) and pro-apoptotic members of mimetic-induced apoptosis, even in the absence of OPA1 the BCL-2 family. The released pro-apoptotic proteins (Fig. 5e), thus placing DRP-1 downstream of OPA1 pro- could then activate the effector proteins (BAK, in H1299 teolysis but upstream of cytochrome c release in BH3 as these cells lack BAX) to oligomerise on mitochondrial mimetic-mediated apoptosis. membranes to subsequently release cytochrome c (Fig. 6a). Fig. 6 DRP-1 regulates BH3 mimetic-induced activation of BAK. a Scheme demonstrating the primary mechanism of action of BH3 mimetics and the downstream events that culminate in apoptosis. b Immunoprecipitation of BCL-X and MCL-1 was carried out in H1299 cells, transfected with control or DRP-1 siRNA, followed by exposure to Z-VAD.fmk (30 µM) for 0.5 h and a combination of A-1331852 (100 nM) and A-1210477 (10 µM) for 4 h, and the eluted complexes were immunoblotted for the indicated proteins. c H1299 cells were treated as b and the extent of BAK activation was assessed by flow cytometry from at least three independent experiments. Error bars = mean ± SEM. Statistical analysis was conducted by one-way ANOVA (***P ≤ 0.001). d Western blots of different molecular weight fractions from size exclusion chromatography showing BAK oligomerisation in H1299 cells upon exposure to A-1331852 (100 nM) and A-1210477 (10 µM) for 2 h. e Immunoprecipitation of active BAK in H1299 cells treated as b, and the eluted complexes were immunoblotted for the indicated proteins. f Representative images of cells showing BAK activation and DRP-1 distribution in H1299 cells treated as b. Scale bar: 10 µm Official journal of the Cell Death Differentiation Association Milani et al. Cell Death Discovery (2019) 5:117 Page 8 of 11 Immunoprecipitation of BCL-X and MCL-1 to identify ascertain whether such fission was a prerequisite for their associated pro-apoptotic proteins revealed that in apoptosis. This difficulty was partly because DRP-1 H1299 cells, BIM and BAK were bound to both BCL-X appeared to play important but distinct roles both in A- and MCL-1, whereas NOXA and BAD exclusively bound 1210477-mediated mitochondrial fission and BH3 to MCL-1 and BCL-X , respectively (Fig. 6b). Exposure of mimetic-mediated apoptosis . Moreover, DRP-1 also these cells to a combination of A-1331852 and A-1210477 interacted with MCL-1 and BCL-X , thus coupling 23,27,36 resulted in displacement of most of these pro-apoptotic mitochondrial fission and apoptosis . However, with proteins from their corresponding ant-apoptotic partners the development of more potent inhibitors, such as (Fig. 6b). Importantly, none of these interactions/dis- S63845, we have demonstrated that mitochondrial fission placements were altered in cells following DRP-1 down- does not occur at concentrations sufficient to inhibit regulation, thus suggesting that DRP-1 played no role in MCL-1 (Fig. 3). Furthermore, while mitochondrial fission the early events of BH3 mimetic-mediated apoptosis. Since induced by A-1210477 and high concentrations of S63845 BAK and other BH3-only proteins were released following was mediated by DRP-1, mitochondrial swelling that BH3 mimetics, we next wished to assess if BAK activation occurred at the onset of apoptosis induction was largely was altered in the absence of DRP-1. Downregulation of independent of DRP-1 (Figs. 3 and 4), thus differentiating DRP-1 resulted in a significant decrease in BH3 mimetic- the distinct types of mitochondrial fission. mediated activation of BAK (Fig. 6c), suggesting that DRP- Our data in the HCT-116 WT and BAX/BAK DKO 1 was critical in the activation of BAK during BH3 cells convincingly demonstrate that BH3 mimetic- mimetic-mediated apoptosis. Although the requirement of mediated OMM breaks and swelling of matrix compart- DRP-1 for BAK activation could be demonstrated, no ment are essential for BAX/BAK to facilitate cytochrome binding of DRP-1 to the oligomerised/active BAK was c release (Fig. 2). The inability of HCT-116 OctaKO cells observed in these cells (Fig. 6d–f), thus suggesting the to prevent BH3 mimetic-mediated mitochondrial changes involvement of other protein(s) in BAK activation imme- further supports our findings that BAX and BAK but not diately preceding cytochrome c release. Taken together, the known BH3-only members are critical for BH3 33,34 our data confirm that DRP-1 plays a critical role at the mimetic-mediated apoptosis . How BAX and BAK level of BAK activation, facilitating OMM breaks, cyto- localise to the sites of OMM breaks to facilitate cyto- chrome c release and apoptosis. chrome c release is not entirely known. The involvement of DRP-1, Dynamin-2, and even membranes of the endoplasmic reticulum in these events have been pre- Discussion 37–42 BH3 mimetics, in particular ABT-737 and ABT-199, viously proposed . Downregulation of DRP-1 or its induce a novel paradigm of cell death, characterised by receptors, MID49 and MID51, have been shown to excessive swelling of mitochondrial matrix and dis- antagonise cytochrome c release and apoptosis in continuities in the OMM in BCL-2-dependent chronic response to a wide variety of apoptotic stimuli . DRP-1 19,31 lymphocytic leukaemia cells . BH3 mimetics targeting functions downstream of OPA1-mediated cristae remo- BCL-X and MCL-1 also induce similar mitochondrial delling (Fig. 5), to activate BAK (Fig. 6), which in turn ultrastructural changes in cells that exclusively depend on precedes BAK oligomerisation and membrane insertion BCL-X and MCL-1, respectively (Fig. 1) . However, cells for the execution of MOMP and apoptosis. However, exposed to the MCL-1 inhibitor, A-1210477, exhibit mitochondrial cristae remodelling requires the presence marked mitochondrial changes, in particular mitochon- of BAX and BAK (Fig. 2) . Thus DRP-1 could function drial fission, irrespective of their dependencies on a spe- either downstream or independent of OPA1 proteolysis to cific BCL-2 family member for survival (Fig. 3). This is in activate BAK and ensuing apoptosis. Taken together, our agreement with our previous findings . Mitochondrial data suggest that BH3 mimetics most likely activate BAX/ fission mediated by A-1210477 alone did not result in BAK independently of the eight known BH3-only mem- apoptosis in these cell lines, even after prolonged expo- bers, which further results in OPA1-mediated cristae sure . This was most probably because most cell lines remodelling to redistribute cytochrome c within the derived from solid tumours depend on both BCL-X and mitochondria, thus priming the mitochondria to undergo MCL-1 for survival, and inhibition of MCL-1 alone was MOMP, upon sensing the stress signal. DRP-1 plays a not sufficient to result in apoptosis. This was further critical role at this stage to activate BAK and/or BAX to supported by our observation that inhibition of MCL-1 insert these effector proteins on mitochondrial mem- using A-1210477 while resulting in extensive mitochon- branes. This along with the constriction of the primed drial fission did not induce OMM breaks and cell death, mitochondria by DRP-1 constitute the so-called stress unless the activity of BCL-X was also neutralised. signals that cause OMM breaks, efficiently releasing the Although A-1210477-mediated mitochondrial fission redistributed cytochrome c into the cytosol and initiating did not necessarily result in apoptosis, it was difficult to apoptosis. Official journal of the Cell Death Differentiation Association Milani et al. Cell Death Discovery (2019) 5:117 Page 9 of 11 Materials and methods metal staining, which consisted of two consecutive Cell culture osmium tetroxide steps (2% (w/v) OsO4 in ddh O), fol- H1299 (purchased from ATCC), K562 (provided by lowed by 1% (w/v) aqueous uranyl acetate. To prevent Prof. R. Clark, University of Liverpool) and MAVER-1 precipitation artefacts, the cells were washed copiously cells (provided by Dr. J. Slupsky, University of Liverpool) with ddH O between each staining step. All fixation and were cultured in RPMI 1640 medium (Life Technologies). staining steps were performed in a Pelco Biowave®Pro H929 cells (purchased from DMSZ, Braunshweig, (Ted Pella Inc., Redding, California, USA) at 100w 20Hg, Germany) were cultured in RPMI 1640 medium supple- for 3 min and 1 min, respectively. Dehydration was in a mented with 0.05 mM β-mercaptoethanol (BME). Colon graded ethanol series before filtration and embedding in cancer cell lines HCT-116 (wild-type and DKO) (from R. medium premix resin (TAAB, Reading, UK). Seventy to J. Youle, National Institute of Health, USA) and HCT- 74 nm serial sections were cut using a UC6 ultra micro- 116-OctaKO were cultured in McCoy’s 5A Modified tome (Leica Microsystems, Wetzlar, Germany) and col- media. All culture media were supplemented with 10% lected on Formvar (0.25% (w/v) in chloroform (TAAB, FBS (Life Technologies) and maintained at 37 °C in a Reading, UK) coated Gilder 200 mesh copper grids humidified atmosphere of 5% CO . All cell lines used in (GG017/C; TAAB, Reading, UK). Images were acquired this study were subjected to short tandem repeat (STR) on a 120 kV Tecnai G2 Spirit BioTWIN (FEI, Hillsboro, profiling to ensure quality and integrity. Oregon, USA) using a MegaView III camera and analySIS software (Olympus, Germany). For immunocytochem- Reagents istry, cells grown on coverslips were fixed with 4% (w/v) ABT-199, A-1210477 and Z-VAD.FMK from Selleck paraformaldehyde, permeabilised with 0.5% (v/v) Triton (Houston, TX, USA), S63845 from Active Biochem X-100 in PBS, followed by incubations with primary (Kowloon, Hong Kong) and A-1331852 from AbbVie Inc. antibodies (diluted 1:250 in 3% BSA in PBS), the appro- (North Chicago, IL, USA) were used. Antibodies against priate fluorophore-conjugated secondary antibodies HSP70 (cat#ab2799) from Abcam (Cambridge, UK), (diluted 1:1000 in 3% BSA in PBS), mounted on glass OPA1 (cat#612607), cytochrome c (cat#556432) and slides and imaged using a 3i Marianas spinning disk DRP-1 (cat#611113) from BD Biosciences (San Jose, CA, confocal microscope, fitted with a Plan-Apochromat ×63/ USA); BCL-X (cat#2762), BIM (cat#2933) and BAD 1.4 NA oil objective, M27 and a Hamamatsu ORCA- (cat#9292) from Cell Signalling Technology (MA, USA); Flash4.0 v2 sCMOS Camera (all from Intelligent Imaging BAK (AB-1) (cat#AM-03) and NOXA (cat#OP180) from Innovations, GmbH, Germany). Millipore (Watford, UK) and MCL-1 (cat#sc-819), BAK (cat#sc-832) and GAPDH (cat#sc-25778) from Santa Cruz Cytochrome c release assay Biotechnologies (Santa Cruz, CA, USA) were used. All Approximately 10 cells were washed in cold PBS and other reagents were obtained from Sigma Aldrich (St. resuspended in mitochondrial isolation buffer (250 mM Louis, MO, USA). sucrose, 20 mM HEPES, pH 7.4, 5 mM MgCl and 10 mM KCl) containing 0.01% digitonin. Cells were left on ice Overexpression and genetic silencing for 5 min followed by centrifugation at 13000 g for 3 min For transient overexpression studies, cells were trans- at 4 °C. Subsequently, the supernatant (cytosolic fraction) fected with GFP-DRP1 K38A plasmid (provided by Dr. E. and pellet (mitochondrial fraction) were processed for Bampton, University of Leicester, UK), using TransIT-LT- western blotting. 1 transfection reagent (Mirus Bio LLC, Madison, WI, USA), according to the manufacturer’s protocol. For RNA Size exclusion chromatography, immunoprecipitation and interference, cells were transfected with 10 nM of siRNAs western blotting against DRP-1 (s104274235), MCL-1 (s8585 or Size exclusion chromatography and immunoprecipi- SI02781205) or BCL-X siRNA (s1920) purchased from tation experiments were carried out as previously 16,27 Qiagen Ltd (Manchester, UK) or ThermoFisher Scientific described . Western blotting was carried out (Waltham, MA, USA). Cells were transfected using 0.33% according to standard protocols. Briefly, 50 μgoftotal (v/v) Interferin reagent (Polyplus Transfection Inc., NY) protein lysate was subjected to SDS-PAGE electro- to culture media, according to the manufacturer’s proto- phoresis. Subsequently proteins were transferred to col and processed 72 h after transfection. nitrocellulose membrane, probed with appropriate pri- mary antibodies (diluted 1:1000 in Tris-buffered saline Microscopy with 0.1% Tween-20), species-specific secondary anti- For electron microscopy, cells were fixed in 2.5% (w/v) bodies (diluted 1:2000 in Tris-buffered saline with 0.1% glutaraldehyde and 2 mM calcium chloride in 0.1 M Tween-20) and protein bands visualised with ECL cacodylate buffer (pH 7.4). This was followed by heavy reagents (GE Healthcare). Official journal of the Cell Death Differentiation Association Milani et al. Cell Death Discovery (2019) 5:117 Page 10 of 11 Received: 5 June 2019 Revised: 17 June 2019 Accepted: 23 June 2019 Flow cytometry The extent of apoptosis in cells following different treatments was quantified by using an Attune NxT flow cytometer (ThermoFisher Scientific, Paisley, UK) follow- References ing staining of the cells with AnnexinV-FITC and propi- 1. 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Cell Death Discovery – Springer Journals

Published: Jul 17, 2019

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