Mitochondrially targeted tamoxifen in patients with metastatic solid tumours: an open-label, phase I/Ib single-centre trial.

Background: Mitochondria present an emerging target for cancer treatment. We have investigated the effect of mitochondrially targeted tamoxifen (MitoTam), a first-in-class anti-cancer agent, in patients with solid metastatic tumours. Methods: MitoTam was tested in an open-label, single-centre (Department of Oncology, General Faculty Hospital, Charles University, Czech Republic), phase I/Ib trial in metastatic patients with various malignancies and terminated oncological therapies. In total, 75 patients were enrolled between May 23, 2018 and July 22, 2020. Phase I evaluated escalating doses of MitoTam in two therapeutic regimens using the 3 + 3 design to establish drug safety and maximum tolerated dose (MTD). In phase Ib, three dosing regimens were applied over 8 and 6 weeks to evaluate long-term toxicity of MitoTam as the primary objective and its anti-cancer effect as a secondary objective. This trial was registered with the European Medicines Agency under EudraCT 2017-004441-25. Findings: In total, 37 patients were enrolled into phase I and 38 into phase Ib. In phase I, the initial application of MitoTam via peripheral vein indicated high risk of thrombophlebitis, which was avoided by central vein administration. The highest dose with acceptable side effects was 5.0 mg/kg. The prevailing adverse effects (AEs) in phase I were neutropenia (30%), anaemia (30%) and fever/hyperthermia (30%), and in phase Ib fever/hyperthermia (58%) together with anaemia (26%) and neutropenia (16%). Serious AEs were mostly related to thromboembolic (TE) complications that affected 5% and 13% of patients in phase I and Ib, respectively. The only statistically significant AE related to MitoTam treatment was anaemia in phase Ib (p = 0.004). Of the tested regimens weekly dosing with 3.0 mg/kg for 6 weeks afforded the best safety profile with almost all being grade 1 (G1) AEs. Altogether, five fatalities occurred during the study, two of them meeting criteria for Suspected Unexpected Serious Adverse Events Reporting (SUSAR) (G4 thrombocytopenia and G5 stroke). MitoTam showed benefit evaluated as clinical benefit rate (CBR) in 37% patients with the largest effect in renal cell carcinoma (RCC) where four out of six patients reached disease stabilisation (SD), one reached partial response (PR) so that in total, five out of six (83%) patients showed CBR. Interpretation: In this study, the MTD was established as 5.0 mg/kg and the recommended dose of MitoTam as 3.0 mg/kg given once per week via central vein with recommended preventive anti-coagulation therapy. The prevailing toxicity included haematological AEs, hyperthermia/fever and TE complications. One fatal stroke and non-fatal G4 thrombocytopenia were recorded. MitoTam showed high efficacy against RCC. Funding: Smart Brain Ltd. Translation: For the Czech translation of the abstract see Supplementary Materials section.

Mitochondria on the move: Horizontal mitochondrial transfer in disease and health.

Mammalian genes were long thought to be constrained within somatic cells in most cell types. This concept was challenged recently when cellular organelles including mitochondria were shown to move between mammalian cells in culture via cytoplasmic bridges. Recent research in animals indicates transfer of mitochondria in cancer and during lung injury in vivo, with considerable functional consequences. Since these pioneering discoveries, many studies have confirmed horizontal mitochondrial transfer (HMT) in vivo, and its functional characteristics and consequences have been described. Additional support for this phenomenon has come from phylogenetic studies. Apparently, mitochondrial trafficking between cells occurs more frequently than previously thought and contributes to diverse processes including bioenergetic crosstalk and homeostasis, disease treatment and recovery, and development of resistance to cancer therapy. Here we highlight current knowledge of HMT between cells, focusing primarily on in vivo systems, and contend that this process is not only (patho)physiologically relevant, but also can be exploited for the design of novel therapeutic approaches.

Mitochondrial Function, Fatty Acid Metabolism, and Body Composition in the Hyperbilirubinemic Gunn Rat.

Background: Circulating bilirubin is associated with reduced adiposity in human and animal studies. A possible explanation is provided by in vitro data that demonstrates that bilirubin inhibits mitochondrial function and decreases efficient energy production. However, it remains unclear whether hyperbilirubinemic animals have similar perturbed mitochondrial function and whether this is important for regulation of energy homeostasis. Aim: To investigate the impact of unconjugated hyperbilirubinemia on body composition, and mitochondrial function in hepatic tissue and skeletal muscle. Materials and Methods: 1) Food intake and bodyweight gain of 14-week old hyperbilirubinemic Gunn (n = 19) and normobilirubinemic littermate (control; n = 19) rats were measured over a 17-day period. 2) Body composition was determined using dual-energy X-ray absorptiometry and by measuring organ and skeletal muscle masses. 3) Mitochondrial function was assessed using high-resolution respirometry of homogenized liver and intact permeabilized extensor digitorum longus and soleus fibers. 4) Liver tissue was flash frozen for later gene (qPCR), protein (Western Blot and citrate synthase activity) and lipid analysis. Results: Female hyperbilirubinemic rats had significantly reduced fat mass (Gunn: 9.94 ± 5.35 vs. Control: 16.6 ± 6.90 g, p < 0.05) and hepatic triglyceride concentration (Gunn: 2.39 ± 0.92 vs. Control: 4.65 ± 1.67 mg g-1, p < 0.01) compared to normobilirubinemic controls. Furthermore, hyperbilirubinemic rats consumed fewer calories daily (p < 0.01) and were less energetically efficient (Gunn: 8.09 ± 5.75 vs. Control: 14.9 ± 5.10 g bodyweight kcal-1, p < 0.05). Hepatic mitochondria of hyperbilirubinemic rats demonstrated increased flux control ratio (FCR) via complex I and II (CI+II) (Gunn: 0.78 ± 0.16 vs. Control: 0.62 ± 0.09, p < 0.05). Similarly, exogenous addition of 31.3 or 62.5 µM unconjugated bilirubin to control liver homogenates significantly increased CI+II FCR (p < 0.05). Hepatic PGC-1α gene expression was significantly increased in hyperbilirubinemic females while FGF21 and ACOX1 was significantly greater in male hyperbilirubinemic rats (p < 0.05). Finally, hepatic mitochondrial complex IV subunit 1 protein expression was significantly increased in female hyperbilirubinemic rats (p < 0.01). Conclusions: This is the first study to comprehensively assess body composition, fat metabolism, and mitochondrial function in hyperbilirubinemic rats. Our findings show that hyperbilirubinemia is associated with reduced fat mass, and increased hepatic mitochondrial biogenesis, specifically in female animals, suggesting a dual role of elevated bilirubin and reduced UGT1A1 function on adiposity and body composition.

Mitocans Revisited: Mitochondrial Targeting as Efficient Anti-Cancer Therapy.

Mitochondria are essential cellular organelles, controlling multiple signalling pathways critical for cell survival and cell death. Increasing evidence suggests that mitochondrial metabolism and functions are indispensable in tumorigenesis and cancer progression, rendering mitochondria and mitochondrial functions as plausible targets for anti-cancer therapeutics. In this review, we summarised the major strategies of selective targeting of mitochondria and their functions to combat cancer, including targeting mitochondrial metabolism, the electron transport chain and tricarboxylic acid cycle, mitochondrial redox signalling pathways, and ROS homeostasis. We highlight that delivering anti-cancer drugs into mitochondria exhibits enormous potential for future cancer therapeutic strategies, with a great advantage of potentially overcoming drug resistance. Mitocans, exemplified by mitochondrially targeted vitamin E succinate and tamoxifen (MitoTam), selectively target cancer cell mitochondria and efficiently kill multiple types of cancer cells by disrupting mitochondrial function, with MitoTam currently undergoing a clinical trial.

Marizomib suppresses triple-negative breast cancer via proteasome and oxidative phosphorylation inhibition.

Purpose: Lacking effective targeted therapies, triple-negative breast cancer (TNBCs) is highly aggressive and metastatic disease, and remains clinically challenging breast cancer subtype to treat. Despite the survival dependency on the proteasome pathway genes, FDA-approved proteasome inhibitors induced minimal clinical response in breast cancer patients due to weak proteasome inhibition. Hence, developing effective targeted therapy using potent proteasome inhibitor is required. Methods: We evaluated anti-cancer activity of a potent proteasome inhibitor, marizomib, in vitro using breast cancer lines and in vivo using 4T1.2 murine syngeneic model, MDA-MB-231 xenografts, and patient-derived tumor xenografts. Global proteome profiling, western blots, and RT-qPCR were used to investigate the mechanism of action for marizomib. Effect of marizomib on lung and brain metastasis was evaluated using syngeneic 4T1BR4 murine TNBC model in vivo. Results: We show that marizomib inhibits multiple proteasome catalytic activities and induces a better anti-tumor response in TNBC cell lines and patient-derived xenografts alone and in combination with the standard-of-care chemotherapy. Mechanistically, we show that marizomib is a dual inhibitor of proteasome and oxidative phosphorylation (OXPHOS) in TNBCs. Marizomib reduces lung and brain metastases by reducing the number of circulating tumor cells and the expression of genes involved in the epithelial-to-mesenchymal transition. We demonstrate that marizomib-induced OXPHOS inhibition upregulates glycolysis to meet the energetic demands of TNBC cells and combined inhibition of glycolysis with marizomib leads to a synergistic anti-cancer activity. Conclusions: Our data provide a strong rationale for a clinical evaluation of marizomib in primary and metastatic TNBC patients.

Selective elimination of senescent cells by mitochondrial targeting is regulated by ANT2.

Cellular senescence is a form of cell cycle arrest that limits the proliferative potential of cells, including tumour cells. However, inability of immune cells to subsequently eliminate senescent cells from the organism may lead to tissue damage, inflammation, enhanced carcinogenesis and development of age-related diseases. We found that the anticancer agent mitochondria-targeted tamoxifen (MitoTam), unlike conventional anticancer agents, kills cancer cells without inducing senescence in vitro and in vivo. Surprisingly, it also selectively eliminates both malignant and non-cancerous senescent cells. In naturally aged mice treated with MitoTam for 4 weeks, we observed a significant decrease of senescence markers in all tested organs compared to non-treated animals. Mechanistically, we found that the susceptibility of senescent cells to MitoTam is linked to a very low expression level of adenine nucleotide translocase-2 (ANT2), inherent to the senescent phenotype. Restoration of ANT2 in senescent cells resulted in resistance to MitoTam, while its downregulation in non-senescent cells promoted their MitoTam-triggered elimination. Our study documents a novel, translationally intriguing role for an anticancer agent targeting mitochondria, that may result in a new strategy for the treatment of age-related diseases and senescence-associated pathologies.

Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells.

Cancer cells without mitochondrial DNA (mtDNA) do not form tumors unless they reconstitute oxidative phosphorylation (OXPHOS) by mitochondria acquired from host stroma. To understand why functional respiration is crucial for tumorigenesis, we used time-resolved analysis of tumor formation by mtDNA-depleted cells and genetic manipulations of OXPHOS. We show that pyrimidine biosynthesis dependent on respiration-linked dihydroorotate dehydrogenase (DHODH) is required to overcome cell-cycle arrest, while mitochondrial ATP generation is dispensable for tumorigenesis. Latent DHODH in mtDNA-deficient cells is fully activated with restoration of complex III/IV activity and coenzyme Q redox-cycling after mitochondrial transfer, or by introduction of an alternative oxidase. Further, deletion of DHODH interferes with tumor formation in cells with fully functional OXPHOS, while disruption of mitochondrial ATP synthase has little effect. Our results show that DHODH-driven pyrimidine biosynthesis is an essential pathway linking respiration to tumorigenesis, pointing to inhibitors of DHODH as potential anti-cancer agents.

Liposomal Delivery of miR-34b-5p Induced Cancer Cell Death in Thyroid Carcinoma.

This study aims to determine the functional roles of microRNA-34b-5p (miR-34b) in the suppression of anaplastic thyroid carcinoma. We used hydration-of-freeze-dried-matrix (HFDM) formulated liposomes (liposome-loaded miR-34b) for effective delivery of miR-34b to anaplastic thyroid carcinoma in vitro and in vivo. Real time polymerase chain was used to determine the level of miR-34b. Immunocytochemistry, Western blot and ELISA were carried out to determine the effect of this manipulation on VEGF-A expression. In addition, an in vivo xenotransplantation mouse model was used to investigate the functional roles of overexpression of miR-34b in the carcinoma. In anaplastic thyroid carcinoma cells, miR-34b expression was low and significant overexpression (p < 0.05) was noted following transfection with liposome-loaded miR-34b. The miR-34b overexpressed thyroid carcinoma cell lines showed reduction in VEGF-A protein expression, decreased cell proliferation, decreased wound healing, reduced cell cycle progression and increased apoptosis (p < 0.05). In in vivo experiments, when compared to control groups, smaller tumours formed upon intravenous administration of liposome-loaded miR-34b. To conclude, the current study confirmed the tumour suppressor properties of miR-34b via VEGF-A regulation in anaplastic thyroid carcinoma. In addition, delivery of miR-34b using cationic liposome could be a useful therapeutic strategy for targeting therapy in the carcinoma.

Alternative assembly of respiratory complex II connects energy stress to metabolic checkpoints.

Cell growth and survival depend on a delicate balance between energy production and synthesis of metabolites. Here, we provide evidence that an alternative mitochondrial complex II (CII) assembly, designated as CIIlow, serves as a checkpoint for metabolite biosynthesis under bioenergetic stress, with cells suppressing their energy utilization by modulating DNA synthesis and cell cycle progression. Depletion of CIIlow leads to an imbalance in energy utilization and metabolite synthesis, as evidenced by recovery of the de novo pyrimidine pathway and unlocking cell cycle arrest from the S-phase. In vitro experiments are further corroborated by analysis of paraganglioma tissues from patients with sporadic, SDHA and SDHB mutations. These findings suggest that CIIlow is a core complex inside mitochondria that provides homeostatic control of cellular metabolism depending on the availability of energy.

Horizontal transfer of whole mitochondria restores tumorigenic potential in mitochondrial DNA-deficient cancer cells.

Recently, we showed that generation of tumours in syngeneic mice by cells devoid of mitochondrial (mt) DNA (ρ0 cells) is linked to the acquisition of the host mtDNA. However, the mechanism of mtDNA movement between cells remains unresolved. To determine whether the transfer of mtDNA involves whole mitochondria, we injected B16ρ0 mouse melanoma cells into syngeneic C57BL/6Nsu9-DsRed2 mice that express red fluorescent protein in their mitochondria. We document that mtDNA is acquired by transfer of whole mitochondria from the host animal, leading to normalisation of mitochondrial respiration. Additionally, knockdown of key mitochondrial complex I (NDUFV1) and complex II (SDHC) subunits by shRNA in B16ρ0 cells abolished or significantly retarded their ability to form tumours. Collectively, these results show that intact mitochondria with their mtDNA payload are transferred in the developing tumour, and provide functional evidence for an essential role of oxidative phosphorylation in cancer.

Mitochondrial Complex II: At the Crossroads.

Mitochondrial complex II (CII), also called succinate dehydrogenase (SDH), is a central purveyor of the reprogramming of metabolic and respiratory adaptation in response to various intrinsic and extrinsic stimuli and abnormalities. In this review we discuss recent findings regarding SDH biogenesis, which requires four known assembly factors, and modulation of its enzymatic activity by acetylation, succinylation, phosphorylation, and proteolysis. We further focus on the emerging role of both genetic and epigenetic aberrations leading to SDH dysfunction associated with various clinical manifestations. This review also covers the recent discovery of the role of SDH in inflammation-linked pathologies. Conceivably, SDH is a potential target for several hard-to-treat conditions, including cancer, that remains to be fully exploited.

Selective Disruption of Respiratory Supercomplexes as a New Strategy to Suppress Her2high Breast Cancer.

AIMS: Expression of the HER2 oncogene in breast cancer is associated with resistance to treatment, and Her2 may regulate bioenergetics. Therefore, we investigated whether disruption of the electron transport chain (ETC) is a viable strategy to eliminate Her2high disease. RESULTS: We demonstrate that Her2high cells and tumors have increased assembly of respiratory supercomplexes (SCs) and increased complex I-driven respiration in vitro and in vivo. They are also highly sensitive to MitoTam, a novel mitochondrial-targeted derivative of tamoxifen. Unlike tamoxifen, MitoTam efficiently suppresses experimental Her2high tumors without systemic toxicity. Mechanistically, MitoTam inhibits complex I-driven respiration and disrupts respiratory SCs in Her2high background in vitro and in vivo, leading to elevated reactive oxygen species production and cell death. Intriguingly, higher sensitivity of Her2high cells to MitoTam is dependent on the mitochondrial fraction of Her2. INNOVATION: Oncogenes such as HER2 can restructure ETC, creating a previously unrecognized therapeutic vulnerability exploitable by SC-disrupting agents such as MitoTam. CONCLUSION: We propose that the ETC is a suitable therapeutic target in Her2high disease. Antioxid. Redox Signal. 26, 84-103.

Mitochondrial Targeting of Metformin Enhances Its Activity against Pancreatic Cancer.

Pancreatic cancer is one of the hardest-to-treat types of neoplastic diseases. Metformin, a widely prescribed drug against type 2 diabetes mellitus, is being trialed as an agent against pancreatic cancer, although its efficacy is low. With the idea of delivering metformin to its molecular target, the mitochondrial complex I (CI), we tagged the agent with the mitochondrial vector, triphenylphosphonium group. Mitochondrially targeted metformin (MitoMet) was found to kill a panel of pancreatic cancer cells three to four orders of magnitude more efficiently than found for the parental compound. Respiration assessment documented CI as the molecular target for MitoMet, which was corroborated by molecular modeling. MitoMet also efficiently suppressed pancreatic tumors in three mouse models. We propose that the novel mitochondrially targeted agent is clinically highly intriguing, and it has a potential to greatly improve the bleak prospects of patients with pancreatic cancer. Mol Cancer Ther; 15(12); 2875-86. ©2016 AACR.

The Assembly Factor SDHAF2 Is Dispensable for Flavination of the Catalytic Subunit of Mitochondrial Complex II in Breast Cancer Cells.

Mitochondrial complex II or succinate dehydrogenase (SDH) is at the crossroads of oxidative phosphorylation and the tricarboxylic acid cycle. It has been shown that Sdh5 (SDHAF2/SDH5 in mammals) is required for flavination of the subunit Sdh1 (SDHA in human cells) in yeast. Here we demonstrate that in human breast cancer cells, SDHAF2/SDH5 is dispensable for SDHA flavination. In contrast to yeast, CRISPR-Cas9 nickase-mediated SDHAF2 KO breast cancer cells feature flavinated SDHA and retain fully assembled and functional complex II, as well as normal mitochondrial respiration. Our data show that SDHA flavination is independent of SDHAF2 in breast cancer cells, employing an alternative mechanism.

Correction: Characterisation of Mesothelioma-Initiating Cells and Their Susceptibility to Anti-Cancer Agents.

[This corrects the article DOI: 10.1371/journal.pone.0119549.].

MicroRNA-126 induces autophagy by altering cell metabolism in malignant mesothelioma.

Autophagy favors both cell survival and cancer suppression, and increasing evidence reveals that microRNAs (MIRs) regulate autophagy. Previously we reported that MIR126 is downregulated in malignant mesothelioma (MM). Therefore, we investigated the role of MIR126 in the regulation of cell metabolism and autophagy in MM models. We report that MIR126 induces autophagic flux in MM cells by downregulating insulin receptor substrate-1 (IRS1) and disrupting the IRS1 signaling pathway. This was specific to MM cells, and was not observed in non-malignant cells of mesothelial origin or in MM cells expressing MIR126-insensitive IRS1 transcript. The MIR126 effect on autophagy in MM cells was recapitulated by IRS1 silencing, and antagonized by IRS1 overexpression or antisense MIR126 treatment. The MIR126-induced loss of IRS1 suppressed glucose uptake, leading to energy deprivation and AMPK-dependent phosphorylation of ULK1. In addition, MIR126 stimulated lipid droplet accumulation in a hypoxia-inducible factor-1α (HIF1α)-dependent manner. MIR126 also reduced pyruvate dehydrogenase kinase (PDK) and acetyl-CoA-citrate lyase (ACL) expression, leading to the accumulation of cytosolic citrate and paradoxical inhibition of pyruvate dehydrogenase (PDH) activity. Simultaneous pharmacological and genetic intervention with PDK and ACL activity phenocopied the effects of MIR126. This suggests that in MM MIR126 initiates a metabolic program leading to high autophagic flux and HIF1α stabilization, incompatible with tumor progression of MM. Consistently, MIR126-expressing MM cells injected into immunocompromised mice failed to progress beyond the initial stage of tumor formation, showing that increased autophagy has a protective role in MM.

Mitochondria: An intriguing target for killing tumour-initiating cells.

Tumour-initiating cells (TICs) play a pivotal role in cancer initiation, metastasis and recurrence, as well as in resistance to therapy. Therefore, development of drugs targeting TICs has become a focus of contemporary research. Mitochondria have emerged as a promising target of anti-cancer therapies due to their specific role in cancer metabolism and modulation of apoptotic pathways. Mitochondria of TICs possess special characteristics, some of which can be utilised to design drugs specifically targeting these cells. In this paper, we will review recent research on TICs and their mitochondria, and introduce drugs that kill these cells by way of mitochondrial targeting.

Combined circulating epigenetic markers to improve mesothelin performance in the diagnosis of malignant mesothelioma.

OBJECTIVES: Malignant mesothelioma (MM) is a highly aggressive tumor with poor prognosis. A major challenge is the development and application of early and highly reliable diagnostic marker(s). Serum biomarkers, such as 'soluble mesothelin-related proteins' (SMRPs), is the most studied and frequently used in MM. However, the low sensitivity of SMRPs for early MM limits its value; therefore, additional biomarkers are required. In this study, two epigenetically regulated markers in MM (microRNA-126, miR-126, and methylated thrombomodulin promoter, Met-TM) were combined with SMRPs and evaluated as a potential strategy to detect MM at an early stage. MATERIALS AND METHODS: A total of 188 subjects, including 45 MM patients, 99 asbestos-exposed subjects, and 44 healthy controls were prospectively enrolled, serum samples collected, and serum levels of SMRPs, miR-126 and Met-TM evaluated. Logistic regression analysis was performed to evaluate the diagnostic value of the three biomarkers. Using this approach, the performance of the '3-biomarker classifier' was tested by calculating the overall probability score of the MM and control samples, respectively, and the ROC curve was generated. RESULTS AND CONCLUSION: The combination of the three biomarkers was the best predictor to differentiate MM patients from asbestos-exposed subjects and healthy controls. The accuracy and cancer specificity was confirmed in a second validation cohort and lung cancer population. We propose that the combination of the two epigenetic biomarkers with SMRPs as a diagnosis for early MM overcomes the limitations of using SMRPs alone.