Mitochondrial Death Research Articles

Correction: Modulating lysosomal function through lysosome membrane permeabilization or autophagy suppression restores sensitivity to cisplatin in refractory non-small-cell lung cancer cells.

[This corrects the article DOI: 10.1371/journal.pone.0184922.].

The Role of Mitochondria in Alzheimer's disease: Neurodegenerative Disease and Future Therapeutic Options

Mitochondria are cytoplasmic organelles responsible for life and death. Extensive evidence from animal and clinical studies suggests that mitochondria play a critical role in aging, cancer, diabetes and neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. Several lines of research suggest that mitochondrial oxidative damage is an important cellular change in most late-onset neurodegenerative diseases. Further, emerging evidence suggests that structural changes in mitochondria, including increased mitochondrial fragmentation and decreased mitochondrial fusion, are critical factors associated with mitochondrial dysfunction and cell death in aging and age-related diseases. In addition, epigenetic factors and lifestyle activities may contribute to selective disease susceptibility for each of these diseases. This paper discusses research that has elucidated features of mitochondria that are associated with cellular dysfunction in aging and neurodegenerative diseases. This paper also discusses mitochondrial abnormalities and potential mitochondrial therapeutics in AD. Alzheimer's disease (AD) is characterized by neuronal loss and gradual cognitive impairment. AD is the leading cause of dementia worldwide and the incidence is increasing rapidly, with diagnoses expected to triple by the year 2050. Impaired cholinergic transmission is a major role player in the rapid deterioration associated with AD, primarily as a result of increased acetylcholinesterase (AChE) in the AD brain, responsible for reducing the amount of acetylcholine (ACh). Current drug therapies, known as AChE inhibitors (AChEIs), target this heightened level of AChE in an attempt to slow disease progression. AChEIs have only showed success in the treatment of mild to moderate AD symptoms, with the glutamate inhibitor memantine being the most common drug prescribed for the management of severe AD.

TRO40303 Ameliorates Alcohol-Induced Pancreatitis Through Reduction of Fatty Acid Ethyl Ester-Induced Mitochondrial Injury and Necrotic Cell Death: Erratum.

TRO40303 Ameliorates Alcohol-Induced Pancreatitis Through Reduction of Fatty Acid Ethyl Ester-Induced Mitochondrial Injury and Necrotic Cell Death: Erratum.

Abstract A06: BRCA-like phenotype as a hallmark of osteosarcoma's converging evolution

Abstract Background: Osteosarcomas are aggressive bone tumors with a high degree of genetic heterogeneity, which has historically limited treatment options in patients who do not respond to standard chemotherapy. Methods: We sequenced exomes and genomes of 32 pretherapeutic osteosarcomas, 6 time-series consisting of chemotherapy-naïve biopsies, resection specimens, recurring tumors and/or metastases, and 15 unpaired metastases from (mainly) poor responders. We then interrogated signatures of mutation processes in cancer genomes and reconciled clonal maps with phylogenetic trees, thus building an evolutionary history of each tumor. Where possible, we searched for evidence of selection acting on the tumors and hallmarks that could be therapeutically exploited. Results: We identified pathogenic mutations in at least 14 cancer drivers, some of which were formerly unknown in the context of osteosarcoma. However, there was no single cancer driver gene that could explain the majority of cases. Instead, >80% of osteosarcomas exhibited a specific combination of single-base substitutions, LOH, or large-scale genome instability characteristic of BRCA1/2-deficient tumors. The BRCA-like phenotype developed through parallel evolution by deleterious mutations affecting the BRCA1/2 genes and their binding partners such as PALB2 or CHEK2. We therefore investigated the sensitivity of a panel of osteosarcoma cell lines to the poly(ADP)-ribose polymerase inhibitor talazoparib alone and in combination with chemotherapeutic drugs. We identified an association between homologous recombination repair deficiency and the response of osteosarcoma cell lines to talazoparib. In combination with temozolomide, talazoparib led to the activation of BAX and BAK, loss of mitochondrial membrane potential, DNA fragmentation, and caspase-dependent cell death. Conclusion: The acquisition of a BRCA-like phenotype through converging evolution has important implications for the development of novel treatment strategies in osteosarcoma using PARP inhibitors alone or together with standard chemotherapy. Citation Format: Michal Kovac, Sebastian Ribi, Claudia Blattmann, Michaela Nathrath, Daniel Baumhoer. BRCA-like phenotype as a hallmark of osteosarcoma's converging evolution [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A06.

Abstract B57: Meclizine synergistically enhances anticancer effects of paclitaxel in A549 lung cancer cells

Abstract Introduction: Lung cancer is the leading cause of cancer-related death worldwide. Despite all improvements in therapy, current standard care for patients with metastatic disease has reached a plateau in the overall response rate. A noteworthy strategy is combining standard anticancer therapy drugs and well-known drugs with novel application in cancer field, such as metformin, statins, and aspirin. This combination is advantageous due to therapeutic efficacy increase, by multiple molecular targeting, and toxicity reduction, by anticancer drug dosage decrease. In this scenario, besides meclizine use on prophylaxis against nausea and vertigo, meclizine was reported as a cellular respiration inhibitor by mechanisms independent of its antihistaminergic and anticholinergic activities. Driven by the importance of mitochondrial activity in cancer, this study aim was to evaluate meclizine anticancer activity and its interaction with placlitaxel in A549 human lung cancer cells. Methods: Meclizine and paclitaxel cytotoxic effects were evaluated in A549 cells by MTT assay. The data were analyzed by Compusyn software to evaluate the drug interaction and the combination index (CI) were determinate. As described by Chou and Talalay classical isobologram equation, CI values greater than 1.1 indicate antagonistic interactions, values between 0.9 and 1.1 indicate additive interactions, and values lower than 0.9 indicate synergistic interactions. Evaluation of apoptotic cells and mitochondrial membrane potential was performed by flow cytometry using Annexin V-Alexafluor488/propidium iodide and tetramethylrhodamine staining, respectively. Proteins expression were evaluated by Western blotting. All experiments were performed in quadruplicate in three independent experiments. All data are expressed as mean ± standard deviation (SD) and were analyzed by one-way ANOVA followed by the Tukey's test using GraphPad Prism software program, version 7. Results: Meclizine was cytotoxic to A549 cells (IC50=275 µM; 95% CI= 255 to 296 µM), leading to mitochondrial membrane depolarization (ratio of 0.35 of median fluorescence intensity reduction vs. control; p<0.001). Indeed, meclizine increased expression of apoptosis-inducing factor (AIF) and cathepsins D and B. In contrast, meclizine triggers a slight apoptosis rate (increase of 13.20% of Annexin V-positive population; p<0.01). Altogether, these results indicated that meclizine induces cell death in caspase-independent manner. Combination Index (CI) evaluation revels that meclizine (200 µM) combined with paclitaxel (40 µM) has stronger cytotoxic effect (CI=0.91). Interestingly, this combination produced an antiproliferative effect greater than the treatments with meclizine (200 µM) or paclitaxel (80 µM) alone. Therefore, meclizine allows a reduction of paclitaxel concentration with an increase of paclitaxel cytotoxic. Overall, these results indicate that in vitro meclizine plus paclitaxel have great cytotoxic effects against A549 cells. Conclusion: Meclizine is cytotoxic to A549 cells, inducing mitochondrial depolarization and caspase-independent cell death. Additionally, meclizine synergistically enhances anticancer effects of paclitaxel in A549 lung cancer cells; therefore, the dosage of paclitaxel can be reduced when associated to meclizine with no efficiency loss. Overall, the combination in vitro of meclizine and paclitaxel is an interesting strategy and may be an effective regimen for lung cancer treatment. Financial support: FAPESP (2015/18528-7 and 2016/09392-7). Citation Format: Sarah Fernandes Teixeira, Ricardo Alexandre De Azevedo, Salomão Dória Jorge, Emer Suavinho Ferro, José Alexandre Marzagão Barbuto, Adilson Kleber Ferreira. Meclizine synergistically enhances anticancer effects of paclitaxel in A549 lung cancer cells [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B57.

Abstract 20933: Regulation of Hypoxia Induced Cardiac Cell Death by the Intrinsic Circadian Clock

Circadian rhythms play a fundamental role in cell metabolism and tissue homeostasis and are influenced by the diurnal oscillation of cellular circadian clock genes. Zeitgebers influence the activation of the circadian rhythm by serving as molecular switches for the intrinsic cellular clock. Herein, we provide new evidence that hypoxia is a Zietgeber for the circadian rhythm and regulator of clock gene expression in cardiac myocytes. We further show that clock gene regulation promotes survival of cardiac myocytes by a mechanism that bi-directionally influences the inducible death protein Bnip3. Cardiac myocytes exhibited phasic oscillations in clock gene expression under basal conditions. Interestingly, in contrast to control cells, a marked time dependent decline in clock gene transcription was observed in cardiac myocytes subjected to hypoxia with maximal decline occurring at 18hrs. This coincided with a reciprocal increase in the expression of the mitochondrial autophagy protein Bnip3 and cell death. Given that Bnip3 is transcriptionally repressed under basal conditions, we reasoned that clock may promote cell survival by negatively regulating Bnip3 gene transcription. While Bnip3 transcription was markedly increased in cardiac myocytes subjected to hypoxia, gain of function of clock suppressed Bnip3 gene activation and cell death of cardiac myocytes. Hence, the findings of the present study provide the first direct evidence that hypoxia is a circadian Zeitgeber for circadian rhythm in cardiac myocytes. We further show that circadian protein Clock regulates cell survival during hypoxic injury by a mechanism that involves the transcriptional repression of Bnip3.

Abstract 1492: Chemical approaches to development of mitochondrial-targeted Hsp90 inhibitor in anti-cancer therapeutics: Mechanism and structure change of mitochondrial Hsp90

Abstract Tumor Necrosis Factor Receptor-Associated Protein 1 (TRAP1), a member of the heat shock protein 90 (Hsp90) which located in mitochondria, is thought to promote tumor cell survival and to reprogram energy metabolism in cancer. Previous studies have demonstrated that the inactivation of TRAP1 selectively induced mitochondrial dysfunction and cell death in cancer cells. Therefore, TRAP1 has been suggested as an emerging target protein for anti-cancer drug development. Isopropyl amine of the Hsp90 inhibitor, PU-H71, was replaced with the mitochondria-targeting moiety, triphenyphosphonium, to produce the mitochondria-accumulating TRAP1 inhibitor, SMTIN-P01. To further optimize SMTIN-P01, we have synthesized several SMTIN-P01 derivatives including TPP-PU-X03 with potent TRAP1 inhibitory activities. TPP-PU-X03 increased TRAP1 ATPase enzyme activity while suppressing chaperone activity, suggesting asymmetric effect on dimeric TRAP1 chaperone functions. Electron microscopic analyses showed TPP-PU-X03 shift TRAP1 conformation from open to close conformation, while other TRAP1 or Hsp90 inhibitors stabilizes open conformation. Importantly, TPP-PU-X03 showed improved mitochondria dysfunction and enhanced cytotoxicity to various cancer cells in vitro and in vivo. Collectively, the structure based optimization of current TRAP1 inhibitors may generate potent anti-cancer drugs with novel mechanisms of action. Citation Format: Sung Hu, Nam Gu Yoon, Byoung Heon Kang. Chemical approaches to development of mitochondrial-targeted Hsp90 inhibitor in anti-cancer therapeutics: Mechanism and structure change of mitochondrial Hsp90 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1492. doi:10.1158/1538-7445.AM2017-1492

Abstract 1497: BPM31510 modulates mitochondrial complex activity to influence oxidative stress in effectuating cell death in multiple cancers

Abstract Dysregulated mitochondria play a multifaceted role in tumorigenesis through regulation of energy production, biomass, redox state, and engagement of cell death pathways. Perturbations in mitochondrial fluxes (i.e inhibition of electron chain complexes activity, impaired electron flow) have substantial effects on cell viability, suggesting that targeting mitochondrial function could be effective for therapeutic response in cancer. BPM31510, containing oxidized coenzyme Q10 elicits an anti-Warburg effect is currently in phase II clinical trials for solid tumors. Previously, we have demonstrated the anti-cancer properties of BPM31510 in breast and pancreatic xenograft models. Here, we examined the mechanism of action of BPM31510 in vitro. Using a multi-cancer cell panel, BPM31510 was shown to be consistently and selectively cytotoxic to cancer cells, compared to normal and non-tumorigenic controls, and sensitivity did not correlate to cell doubling time or mutational status. Treatment with BPM 31510 (EC50) in breast and pancreatic cancer cells resulted in a time- and dose-dependent decrease in mitochondrial membrane potential which preceded an increase in early and late apoptosis cells, suggesting BPM31510 initiates a mitochondrial mediated cell death pathway. Using a fluorescently labeled CoQ10 we were able to trace the subcellular location of the CoQ10, which predominantly accumulates in mitochondria and lipid droplets in a time dependent manner. Additionally, the mitochondrial enrichment of CoQ10 is accompanied by morphological changes that varied amongst the different cancer cell types. As CoQ10 is a redox molecule required for electron transfer activity between complexes, we hypothesized that disruption of Q-pool homeostasis would alter complex activity. To investigate this, Complex driven respiration was measured in cells treated with BPM31510 and compared to untreated. Alterations in mitochondrial respiration characterized by a dose-dependent decrease in succinate (Complex II) and glycerol-3-phosphate (Complex III)-fueled respiration were observed in cells treated with BPM31510, while no changes were seen in pyruvate driven respiration (Complex I), suggesting that BPM31510 specifically impairs respiration responses that are more dependent on Q-pool functionality. As impairment of the electron transport chain increases intracellular oxidative stress, we next investigated if BPM 31510 treatment increases ROS levels. After 24h treatment, BPM31510 significantly increased ROS levels in treated cancer cells compared to untreated. Furthermore, BPM31510 induced death could be in part prevented by co-treatment with antioxidants. Together, these data demonstrates BPM31510 has anti-cancer activity in multiple cancer cell types and define a unique and novel functional link between mitochondrial Q-pool disruption and the mechanism of action of BPM31510. Citation Format: Tulin Dadali, Katerina Krumova, Anne R. Diers, Pallavi Awate, Ryan Ng, Arleide Lee, Stephane Gesta, Vivek K. Vishnudas, Rangaprasad Sarangarajan, Niven R. Narain. BPM31510 modulates mitochondrial complex activity to influence oxidative stress in effectuating cell death in multiple cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1497. doi:10.1158/1538-7445.AM2017-1497

News about VDAC1 in Hypoxia.

The voltage-dependent anion channel (VDAC) is the main interface between the cytosol and mitochondria of cells. It plays a crucial role in both mitochondrial metabolism and cell death. The main basic function of this channel is to mediate and gate the flux of small ions, metabolites, and adenosine triphosphate. Changes in its structure, and thus conformation, are expected to affect its activity and modulate the ability of cancer cells to expand. In this review, we describe a novel mechanism by which mitochondria of cells in hypoxia, a low level of oxygen, protects from apoptosis. In hypoxia, some mitochondria become enlarged due to hyperfusion. These mitochondria possess a truncated form of VDAC1 (VDAC1-ΔC), which is linked to the higher metabolic capacity and the greater resistance to cell death of hypoxic cells. However, not all of the VDAC1 protein is truncated, but the amount of the full-length form is diminished compared to the amount in normoxic cells. First, we describe how such a decrease effects cell proliferation, respiration, glycolysis, and other processes. Second, we report on a novel mitochondrial-endolysosomal crosstalk that leads to VDAC1 truncation. By pharmacological targeting of VDAC1-ΔC, the production of energy could be turned off and the sensitivity to cell death restored. This could counteract the favorable microenvironment that gives cancer cells a growth advantage and thereby disrupts the balance between life and death, which is controlled by VDAC1.

Abstract 375: MCL-1 Regulates Mitochondrial Dynamics and Mitophagy

The anti-apoptotic BCL-2 family protein Myeloid Cell Leukemia-1 (MCL-1) is essential for maintaining mitochondrial integrity and cardiac function in the adult heart. Recently, we reported that cardiac-specific deletion of MCL-1 in mice leads to rapid mitochondrial dysfunction, hypertrophy, and lethal cardiomyopathy. Surprisingly, MCL-1 ablation does not result in apoptotic cell death, but rather cells show signs of mitochondrial deterioration and necrotic cell death. This suggests that in addition to its anti-apoptotic role, MCL-1 has unidentified roles in maintaining mitochondrial function in cardiac myocytes. MCL-1 exists in two distinct locations in myocytes: one form is localized to the outer mitochondrial membrane (MCL-1 OM ) and a shorter cleaved form exists in the mitochondrial matrix (MCL-1 Matrix ). Interestingly, overexpression of MCL-1 WT or MCL-1 OM , but not MCL-1 Matrix , leads to translocation of Drp1 to mitochondria, which correlates with fission and perinuclear aggregation of mitochondria. We also found that Drp1 co-immunoprecipitates with MCL-1 in heart lysates and that MCL-1-deficient hearts lack mitochondrial Drp1. Additionally, the interaction between Drp1 and MCL-1 and mitochondrial fission increase in response to serum starvation in neonatal myocytes. This suggests that MCL-1 OM recruits Drp1 to mitochondria to induce fission. Since there is a strong link between mitochondrial fission and degradation, we examined the effect of MCL-1 on mitophagy. Parkin is an E3 ubiquitin ligase that plays an important role in mitophagy. MEFs, which lack detectable Parkin, do not efficiently clear their depolarized mitochondria in response to FCCP treatment. However, overexpression of Parkin or MCL-1 increased the efficiency of mitophagy in MEFs in response to FCCP treatment. MCL-1-mediated mitophagy is abrogated in autophagy-deficient Atg5 -/- MEFs, confirming that the clearance occurs via enhanced autophagy. Thus, our data suggest that MCL-1 OM has dual actions in coordinating Drp1-mediated fission and mitophagy, which allows for more efficient removal of damaged mitochondria.

Abstract 3731: TSC deficiency unmasks a novel protective role of RIP1/RIP3 signaling against mitochondrial and oxidative stress-induced cell death

Abstract Tuberous sclerosis complex (TSC) is a genetic multi-organ disorder characterized by the development of neoplastic lesions in kidney, lung, brain, heart and skin. It is caused by an inactivating mutation in tumor suppressor genes coding the TSC1/TSC2 complex, resulting in hyperactivation of mTOR- and Raf/MEK/MAPK-dependent signaling that stimulates tumor cell proliferation and metastasis. Despite its oncogenic effect, cells with TSC deficiency were more sensitive to oxidative stress and dependent on mitochondrial metabolism, providing a rationale for a new therapeutic approach. The presented study shows that simultaneous inhibition of two major pathways regulating redox homeostasis using L-Buthionine-sulfoximine (BSO, glutathione synthesis inhibitor) and auranofin (thioredoxin reductase inhibitor) induces oxidative burst, mitochondrial damage and necrotic cell death in TSC2 deficient cells in a highly synergistic and cell context-specific manner. Furthermore, blocking RIP1/RIP3-dependent signaling using chemical inhibitors necrostatin-1 and necrosulfonamide (NSA), reported by others to protect from some cytotoxic stimuli, synergizes with BSO and auranofin in killing TSC2 deficient cells. Expression analysis demonstrated that RIP1 and RIP3 protein levels are elevated in cells with TSC2 deficiency, and their inactivation enhances mitochondrial dysfunction. Finally, supplementation with the mitochondrial metabolite alpha-ketoglutarate, whose synthesis is regulated by RIP1/RIP3, rescues cells from the sensitizing effect of necrostatin-1 and NSA. Together, this study identifies a novel TSC context-dependent role of RIP1/RIP3 signaling in regulating mitochondrial and oxidative homeostasis, and substantiates glutathione, thioredoxin and RIP1/RIP3 pathways targeting as a promising therapeutic approach against TSC-associated tumors. Citation Format: Piotr T. Filipczak, Cindy Thomas, Wenshu Chen, Andrew Salzman, Jacob D. McDonald, Yong Lin, Steven A. Belinsky. TSC deficiency unmasks a novel protective role of RIP1/RIP3 signaling against mitochondrial and oxidative stress-induced cell death. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3731.

Abstract 418: Mitochondrial perturbations as a novel approach to personalized medicine

Abstract Recent progress in cancer research has been marked by the continued development of exciting drugs that selectively target vulnerabilities present only in cancer cells, so-called targeted therapies. Too often, however, we are lacking the proper predictive biomarkers to identify which patients will benefit most from individual targeted therapies. Since most chemotherapeutic agents ultimately function by inducing the mitochondrial apoptotic pathway, we hypothesized that a tool that measures mitochondrial sensitivity may serve as a broadly predictable biomarker. Hence to address the unmet need for predictive biomarkers, we have developed a tool called Dynamic BH3 Profiling (DBP). In this approach, we briefly treat cancer cells (14h) from patients with targeted drugs and measure the apoptotic threshold of a cell “priming” by treatment with peptides derived from BH3 domains of pro-apoptotic proteins. Previous studies from our laboratory demonstrated that when a patient's cells exhibit robust death signaling in the DBP assay in response to a particular drug, it predicts a good clinical response to that drug in patients. Using xenotransplantation of primary AML samples into NSG mice (PDX models), we validated the utility of DBP to personalize acute myeloid leukemia therapy. We established 12 independent AML PDX models and determined the mitochondrial response of splenic myeloblasts from each PDX to 25 targeted agents. Ex-vivo DBP measurement demonstrated heterogeneous responses; while some drugs induced priming in all PDXs, others increased priming in only selective PDX models. For instance, JQ-1 (BRD-4 inhibitor) and birinapant (SMAC mimetic) demonstrated inverse correlation of their activity among 3 different PDXs. These suggested that mitochondrial priming can stratify AML PDX models according to predicted sensitivity to targeted agents. When we compared DBP results with cell death assay, we found that myeloblasts from most of the PDXs failed to sustain long-term culture (72h), a requirement for cytotoxicity measurements. However, myeloblasts from 3 of the PDXs with high starting viability showed strong correlation between mitochondrial priming and cell death, implying that cells with increased mitochondrial response are destined to commit apoptosis. By testing functional state of the apoptotic pathway in myeloblasts from spleen, bone-marrow and peripheral blood with BH3 profiling, we found that splenic blasts were relatively more primed for apoptosis in 8 out of 10 PDXs. Correlatively, drug induced priming also varied between myeloblasts from these 3 different sites. Finally, we are directly testing the performance of DBP as a predictive biomarker by treating a variety of PDX models with drugs, including drugs predicated to be active and drugs predicated to be inactive. Collectively, mitochondria based measurements can predict good and bad response to individual targeted agents and may serve as a powerful biomarker to choose patient therapy. Citation Format: shruti bhatt, david weinstock, Anthony Letai. Mitochondrial perturbations as a novel approach to personalized medicine. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 418.

Abstract 3269: Recapitulating the BETH adjuvant breast cancer trial (NCT00625898) using clinically accurate orthotopic surgical resection models

Abstract Background: An overwhelming majority of phase III clinical trials in the oncology setting fail as promising results observed in the ‘pre-clinic’ are not successfully translated to patients. As such, improved models which more accurately predict outcomes are now required. Herein, we have established orthotopic surgical resection models of Her2+ breast cancer, which replicate the phenotype of clinical post-resection metastasis [1,2]. Using these models we have recapitulated the BETH adjuvant breast cancer trial (NCT00625898) where the addition of bevacizumab (BVZ) to chemotherapy plus trastuzumab (TRAST) failed to provide additional benefit in the adjuvant setting. Methods: SCID mice were orthotopically implanted with bioluminescent Her2+ MDA-MB-231or HCC1954 cells and palpable tumors resected approximately 5 weeks later. 3 weeks after resection, mice were treated with 10 mg/kg TRAST + 5mg/kg paclitaxel IP once weekly for 6 cycles with or without weekly BVZ (5mg/kg IP). Metastasis was monitored by weekly bioluminescence imaging. Results: Tumor growth and imaging data confirmed that the addition of BVZ to adjuvant TRAST + chemotherapy provided no additional benefit compared with TRAST + chemotherapy alone. Previous pre-clinical studies using inappropriate non-resection models failed to predict this response. Furthermore, Reverse Phase Protein Array analysis of treated tumors implicated HER2, VEGF, mTOR and the intrinsic mitochondrial cell death apoptotic pathways in treatment resistance. Conclusion: In summary, our data provides evidence for pre-clinical models which better predict clinical outcome in the breast cancer adjuvant setting, and which represent an important resource for interrogating resistance pathways and identifying novel biomarker signatures. [1]. Breast. 2013 Aug;22 Suppl 2:S57-65; [2]. Cancer Res. 2013 May 1;73(9):2743-8.This work was performed under Irish HPRA Authorization AE18982 and was supported by the Clinical Cancer Research Trust. ATB receives funding from the Irish Cancer Society Collaborative Cancer Research Centre BREAST-PREDICT Grant (CCRC13GAL). Citation Format: Liam S. Shiels, Ian S. Miller, Clare Morgan, Mattia Cremona, Robert Kerbel, Bryan Hennessey, Norma O’Donovan, John Crown, Annette T. Byrne. Recapitulating the BETH adjuvant breast cancer trial (NCT00625898) using clinically accurate orthotopic surgical resection models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3269.

Abstract 4061: Origanum majorana organic extract induces senescence and autophagic cell death in breast cancer cells through influencing mitochondrial metabolism

Abstract Excess 17β estradiol (E2) is a risk factor of breast cancer. Previously, we have reported that E2 through influencing mitochondria by unknown mechanism contribute to the estrogen induced breast carcinogenesis. The aim of this study was to evaluate the mitotoxic and cytotoxic effects of normal O. Majorana organic extract (OME) as well as PEGylated nanoconjugate of OME with triphenylphosphonium (P-OME) against human breast epithelial and cancer cell lines. Origanum majorana, commonly known as marjoram, is a perennial herb, which is widely used in the Middle East as a spice. It has been shown to possess extensive range of biological activity, including antioxidant, anti-inflammatory, and anti-tumor growth effects. Interestingly, the anticancer potential of O. Majorana against breast cancer remains largely unexplored. Here, we investigated the anticancer effect of O. Majorana on three breast cell lines. We also used triphenylphosphonium (TPP) cation to check whether an imbalance in mitochondrial bioenergetics, in part, may be responsible for estrogen induced growth of breast cancer. Determination of cell density (cell survival) by SRB and mitochondrial metabolic activity by MTT assays showed that both OME and P-OME reduced growth of MDA-MB-231 and MCF-7 cells, but no effect on normal breast epithelial MCF-10A cells. OME and TPP blocked E2-induced increases of cell survival, metabolic activity and proliferation of breast cancer MCF-7 cells. E2 treatment increased MitoTracker® Red 580 labeling, indicating E2 treatment increased MCF7 cells mitochondrial mass. We observed a several fold increase in mitochondrial transcription factor A (TFAM) DNA binding activity as early as 3 hrs in treated MCF7 cells. DNA synthesis was inhibited in E2-exposed MCF7 cells by the silencing of TFAM. To discern whether a decrease in ATP production may be responsible for the E2-induced growth signaling, we measured the ATP present in the MCF7 cells. Our data showed that the ATP levels in E2 treated cells were very similar to control cells. E2 treatment of MCF-7 cells increased mRNA and protein levels of BNIP3 involved in mitophagy/autophagy. Together these data suggest that a carcinogenic concentration of E2 may modify mitochondrial dynamics, mitophagy, biogenesis and metabolism. In summary, our results demonstrated for the first time that OME was able to inhibit estrogen-induced growth of MCF-7 cells in a time- and concentration-dependent manner. Our results also demonstrated that P-OME nanoconjugate compared to OME was far more effective in exerting its cytotoxic effect through the induction of growth arrest, mitochondrial metabolic activity, senescence, apoptosis and autophagic cell death in the highly metastatic triple negative MDA-MB-231 cells. Our findings offer a new perspective on the utility of O. Majorana plant extract to be developed as a new alternative medicinal therapy against breast tumors. Citation Format: Mohannad Garoub, Jayanta Das, Stanislaw Wnuk, Deodutta Roy. Origanum majorana organic extract induces senescence and autophagic cell death in breast cancer cells through influencing mitochondrial metabolism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4061.

Abstract 3492: Differential sensitivity of normal liver and hepatocarcinoma tissues to tBid induced apoptosis is determined by the abundance of VDAC2

Abstract A conserved step in extrinsic apoptosis is the outer mitochondrial membrane (OMM) permeabilization by truncated Bid (tBid) that is generated from Bid upon cleavage by Caspase-8 activated at death receptors. tBid directly or indirectly activate proapoptotic proteins i.e. Bak in OMM and/ or Bax in cytosol which is followed by a change in their conformation and oligomerization, leading to a pore formation in the OMM. To better understand the mitochondrial phase of this pathway we applied tBid to semi-permeabilized cells and isolated mitochondria. We monitored cytochrome c release, and the ensuing mitochondrial membrane potential collapse, using immunoblotting and fluorimetry as the markers for mitochondrial cell death. We found that normal liver tissue and normal hepatocytes (Rat, Mouse or Human) are unexpectedly resistant to tBid, whereas hepatocellular carcinoma (HCC) tissues and cell lines (e.g. PLC, Huh7) are greatly sensitized. Considering that the effect of tBid is most effectively mediated through Bak, its abundance in the mitochondria was tested. Bak level is significantly higher in tumor tissue/cell lines, whereas OMM-bound tBid and Bax levels are similar in both cancerous and normal tissue. Since we have recently found that voltage dependent anion channel 2 (V2), a pore forming OMM protein, is specifically required for Bak targeting to OMM, the abundance of V2 was also measured. V2 showed significant upregulation in HCC tissue/cell lines. Rescue studies using V2 sensitized normal hepatocytes and liver mitochondria to tBid. We propose that V2 might be the main regulator for the differential tBid sensitivity in normal liver and hepatocellular carcinoma. Citation Format: Shamim Naghdi, Soumya Sinha Roy, Ludivine Walter, Gyorgy Hajnoczky. Differential sensitivity of normal liver and hepatocarcinoma tissues to tBid induced apoptosis is determined by the abundance of VDAC2. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3492.

PARP-1-modulated AIF translocation is involved in streptomycin-induced cochlear hair cell death

Conclusion SM-induced dose- and location-dependent cochlear hair cell death in vitro. AIF might be translocated from mitochondria to nucleus and cytoplasm within SM-treated hair cells. The translocation of AIF might be modulated by PARP-1.Objective Streptomycin (SM), one of the widely used aminoglycoside nowadays, is still causing significant permanent sensorineural hearing loss owing to sensory hair cell death. This study was designed to investigate the role of apoptosis-inducing factor (AIF), an important mitochondrial cell death regulator, in SM ototoxicity within neonatal rat cochleae and HEI-OC1 cells.Methods The viability of HEI-OC1 cells was quantified by MTT assay. AIF, PARP-1, and myosin VIIa distributions were achieved by immunofluorescence. mRNA and protein expression of AIF and PARP-1 were examined by q-PCR and Western-blot.Results The hair cell loss was concomitant with the SM concentration variation, and aggravated from apical to basal turn. AIF was detected in nuclear region and AIF mRNA was up-regulated after SM incubation. Besides, AIF protein expression in mitochondria was decreased, whereas in cytosol it was increased. PARP-1 mRNA and protein were also up-regulated. 3-AB could attenuate the cell death and reverse the changes of AIF distribution by blocking PARP-1.

Modulation of mitochondrial ion transport by inorganic polyphosphate - essential role in mitochondrial permeability transition pore

Inorganic polyphosphate (polyP) is a biopolymer of phosphoanhydride-linked orthophosphate residues. PolyP is involved in multiple cellular processes including mitochondrial metabolism and cell death. We used artificial membranes and isolated mitochondria to investigate the role of the polyP in mitochondrial ion transport and in activation of PTP. Here, we found that polyP can modify ion permeability of de-energised mitochondrial membranes but not artificial membranes. This permeability was selective for Ba2+ and Ca2+ but not for other monovalent and bivalent cations and can be blocked by inhibitors of the permeability transition pore – cyclosporine A or ADP. Lower concentrations of polyP modulate calcium dependent permeability transition pore opening. Increase in polyP concentrations and elongation chain length of the polymer causes calcium independent swelling in energized conditions. Physiologically relevant concentrations of inorganic polyP can regulate calcium dependent as well calcium independent mitochondrial permeability transition pore opening. This raises the possibility that cytoplasmic polyP can be an important contributor towards regulation of the cell death.

Abstract P5-04-02: The histone deacetylase inhibitor entinostat enhances the efficacy of the MEK inhibitor pimasertib against aggressive types of breast cancer through Noxa-mediated myeloid cell leukemia 1 degradation

Abstract Purpose: Inflammatory breast cancer (IBC) and triple-negative breast cancer (TNBC) are the two most aggressive types of breast cancer whose molecular mechanisms remain unclear, representing a challenge for the development of effective targeted therapies. Single agent targeted therapies are of limited effectiveness in these types of breast cancer. Therefore, we aim to identify new combination therapeutic candidates for these aggressive diseases by comprehensive genomic screening. Experimental Design: We screened kinome siRNA libraries with the mitogen/extracellular signal-regulated kinase (MEK) inhibitor [pimasertib], and genome-wide functional mRNA expression with the histone deacetylase (HDAC) type I inhibitor [entinostat] in TNBC and IBC cell lines. We evaluated the relationship between targets of interest and breast cancer patient survival using the IBC consortium database composed of breast cancer patient samples with clinical follow up. After identifying the targets, we assessed the combinational synergistic effect and its mechanism via cytotoxicity assay, flow cytometry, anchorage-independent growth, quantitative real-time polymerase chain reaction, small interfering RNA, western blotting, and mammary fat pad xenograft mouse models. Results: We identified that knock-down of myeloid cell leukemia 1 (Mcl-1), an anti-apoptotic member of the B-cell lymphoma 2 (Bcl-2) family of apoptosis-regulating proteins, enhanced the anti-proliferative effect of pimasertib. We observed that entinostat induced the expression of Noxa, a pro-apoptotic BH3-only member of the Bcl-2 family that is known to bind and degrade Mcl-1. Interestingly, in a breast cancer patient cohort (N = 389), high Mcl-1/low Noxa co-expression was associated with poorer survival outcomes than low Mcl-1/high Noxa co-expression (P = 0.0038). We found that combination with pimasertib and entinostat enhanced the inhibition of tumor cell proliferation (P < 0.001) compared with entinostat or pimasertib alone. We also observed significant in vivo tumor growth inhibition in both IBC (SUM190, P < 0.0001) and TNBC (SUM149, P < 0.05) xenograft models. Specifically, TNBC and IBC cell lines that overexpressed Noxa after treatment with entinostat were observed to be selectively sensitive to combination treatment with pimasertib. The synergistic antitumor activity of the entinostat- pimasertib combination was due to increased expression of Noxa, which induced the degradation of Mcl-1, resulting in the induction of mitochondrial cell death. Conclusion: Our data provide evidence that entinostat has enhanced antitumor effect in combination with pimasertib, resulting in the induction of apoptosis by Noxa-mediated Mcl-1 degradation. These findings provide a novel preclinical rationale for developing a clinical trial based on combinatorial HDAC and MEK inhibition therapy for TNBC and IBC with high Mcl-1 expression. Citation Format: Torres-Adorno AM, Lee JJ, Kogawa T, Bartholomeusz C, Pitner MK, Ordentlich P, Lim B, Tripathy D, Ueno NT. The histone deacetylase inhibitor entinostat enhances the efficacy of the MEK inhibitor pimasertib against aggressive types of breast cancer through Noxa-mediated myeloid cell leukemia 1 degradation. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-02.

Abstract A88: Openers of voltage dependent anion channels are anti-Warburg agents that enhance mitochondrial metabolism, decrease glycolysis, activate JNK and induce ROS-dependent killing of cancer cells

Abstract Background: Warburg metabolism is characterized by enhanced aerobic glycolysis and suppressed mitochondrial metabolism. Voltage dependent anion channels (VDAC) located in the mitochondrial outer membrane control flux of metabolites into mitochondria. Free α,β-tubulin closes VDAC in planar lipid bilayers (Rostovtseva et al., PNAS 105:18746), and high free tubulin in cancer cells decreases mitochondrial membrane potential (ΔΨ) by limiting ingress of respiratory substrates and ATP that supportΔΨ formation (Maldonado et al., Cancer Res. 70:10192). The small molecule erastin opens VDAC by antagonizing the inhibitory effect of tubulin on VDAC (Maldonado et al., JBC 288:11920). Here, we hypothesized that erastin and erastin-like antagonists of VDAC-tubulin interaction increase mitochondrial redox state, decrease glycolysis, increase mitochondrial formation of reactive oxygen species (ROS) and activate c-jun N-terminal kinase (JNK), culminating in mitochondrial dysfunction and death of cancer cells. Our AIM was to evaluate the effects of erastin/erastin-like compounds on mitochondrialΔΨ, NAD(P)H+ and ROS, lactate generation, JNK activation and cell killing in HepG2 and Huh7 hepatocarcinoma cells. Methods: Confocal/multiphoton fluorescence microscopy assessedΔΨ (tetramethylrhodamine methylester), ROS (chloromethyldichlorofluorescein [cmDCF]; MitoSOX Red) and NAD(P)H (autofluorescence). JNK was assessed by Western blotting and cell killing by propidium iodide fluorometry. Results: In both HepG2 and Huh7, erastin and small molecules X1 and X2 identified in a high-throughput screen increasedΔΨ and NAD(P)H. The three compounds also prevented mitochondrial depolarization by cytosolic high free tubulin induced by nocodazole (microtubule depolymerizing agent). Initial increases ofΔΨ were followed by mitochondrial depolarization occurring 1-2 h after X1 and X2 and 3-4 h after erastin. Lactate generation tested after X1 decreased by 60%. cmDCF and MitoSOX fluorescence increased 30 min after X1 and 60 min after X2 and erastin. The mitochondrially targeted antioxidant MitoQ blocked this increase of ROS. Additionally, erastin caused JNK activation with maximal phosphorylation within 1 h. Both X1 and X2 caused killing of cancer cells (~93% and ~76% respectively), which the antioxidant N-acetylcysteine (100µM) blocked. By contrast, X1 and X2 caused <25% cell death in primary rat hepatocytes. Conclusion: Antagonists of VDAC-tubulin interaction promote mitochondrial metabolism and inhibit glycolysis. These anti-Warburg drugs also cause mitochondrial generation of ROS, which in turn leads to JNK activation, mitochondrial dysfunction and selective death of cancer cells that is prevented by antioxidants. Grants: T32DK083262 to DND; DK073336, DK037034 and 14.Z50.31.0028 to JJL; ACS 13-043-01 and COBRE Pilot Project GM103542 to ENM. Citation Format: David N. DeHart, Monika Gooz, John J. Lemasters, John J. Lemasters, Eduardo N. Maldonado. Openers of voltage dependent anion channels are anti-Warburg agents that enhance mitochondrial metabolism, decrease glycolysis, activate JNK and induce ROS-dependent killing of cancer cells. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A88.

Abstract 16535: Conditional Knockout of the Mitochondrial 18-kDa Translocator Protein Limits Heart Failure

Introduction: Heart failure results in myocyte and mitochondrial death, and is characterized by abnormalities in mitochondrial calcium handling, energy production, and opening of the mitochondrial permeability transition pore (mPTP). The 18-kDa mitochondrial translocator protein (TSPO) has been shown to be significantly upregulated by heart failure in animals and in explanted hearts from patients, suggesting a vital role for this protein. Hypothesis: In the current experiments, we tested the hypothesis that conditional knockout of the TSPO using Cre inducible TSPO-floxed C57BL/6J mice would limit heart failure resulting from transverse aortic constriction (TAC). Methods: Mice in 4 groups_wild-type (WT) sham, WT TAC, KO sham, and KO TAC_were monitored by weekly echocardiography for 8 weeks, followed by downstream experiments. Results: TAC caused a 46±13% reduction in ejection fraction in WT mice, which was significantly lower in the KO mice (14±10%, P < 0.01, Figure). Strain analysis revealed a significant improvement in radial, longitudinal, and circumferential strain in KO TAC mice, supported by tissue histology finding of significantly less collagen in KO TAC mice. KO TAC mice showed lower heart to body weight and lung to body weight ratios, with no KO TAC mice showing signs of pulmonary edema. Calcium uptake experiment using Rhod-2 AM in isolated cells revealed that KO TAC mice had higher mitochondrial calcium uptake, a crucial finding since mitochondrial calcium uptake has been shown to play a role in energetics and mPTP opening. Conclusion: Genetic modulation of the TSPO limits heart failure due to pressure overload, likely mediated by preserving mitochondrial calcium uptake and energy production, and possibly limiting mPTP opening. These data suggest that pharmacologic interventions that inhibit TSPO expression or function can limit heart failure at the sub-cellular level.