Mitochondrial Metabolic Pathways Research Articles

Mito-Specific Nutri-Hijacker Synergizing Mitochondrial Metabolism and Glycolysis Intervention for Enhanced Antitumor Bioenergetic Therapy.

Metabolic rewiring, a dynamic metabolic phenotype switch, confers that tumors exist and proliferate after fitness (or preadaptation) in harsh environmental conditions. Glycolysis deprivation was considered to be a tumor's metabolic Achilles heel. However, metabolic configuration can flexibly retune the mitochondrial metabolic ability when glycolysis is scared, potentially resulting in more aggressive clones. To address the challenge of mitochondrial reprogramming, an antiglycolytic nanoparticle (GRPP NP) containing a novel mitochondrial-targeted reactive oxygen species (ROS) generator (diIR780) was prepared to hijack glucose and regulate mitochondria, thus completely eliminating tumorigenic energy sources. In this process, GRPP NPs@diIR780 can catalyze endogenous glucose, leading to significantly suppressed glycolysis. Moreover, diIR780 can be released and selectively accumulated around mitochondria to generate toxic ROS. These combined effects, in turn, can hamper mitochondrial metabolism pathways, which are crucial for driving tumor progression. This synchronous intervention strategy enables utter devastation of metabolic rewiring, providing a promising regiment to eradicate tumor lesions without recurrence.

MicroRNA 29 modulates β-cell mitochondrial metabolism and insulin secretion via underlying miR-29-OXPHOS complex pathways.

MicroRNAs (miRNAs) regulate β-cell function, and β-cell mitochondria and insulin secretion are perturbed in diabetes. We aimed to identify key miRNAs regulating β-cell mitochondrial metabolism and novel β-cell miRNA-mitochondrial pathways. TargetScan (http://www.targetscan.org/) was used to predict if 16 miRNAs implicated in β-cell function target 27 cis-eGenes implicated in mitochondrial activity. The expression of candidate miRNAs and insulin secretion after 24 and 1 h pre-incubation in 2.8, 11.1- and 16.7-mM glucose was measured in clonal INS-1 832/13 β-cells. MiR-29 silenced INS-1 832/13 cells were assessed for insulin secretion (glucose, pyruvate, and K+), target cis-eGene expression (Ndufv3 and Ndufa10 components of mitochondrial complex I (CI)), OXPHOS (CI-V) protein expression, and mitochondrial OXPHOS respiration/activity. The expression of differentially expressed miR-29 miRNAs was evaluated in Goto-Kakizaki (GK) rat, db/db mouse and type 2 diabetic (T2D) human islets, as well as NMRI mouse islets cultured under glucolipotoxic conditions. MiR-29, miR-15 and miR-124 were predicted to regulate ~20 cis-eGenes, while miR-29 alone was predicted to regulate ≥12 of these in rat and human species. MiR-29 expression and insulin secretion were reduced in INS-1 832/13 cells after 24 h in elevated glucose. MiR-29 knockdown increased all tested insulin secretory responses, Nudfv3, Ndufa10, complex I and II expression, and cellular mitochondrial OXPHOS. MiR-29 expression was reduced in db/db islets but increased in GK rat and T2D human islets. We conclude miR-29 is a key miRNA in regulating β-cell mitochondrial metabolism and insulin secretion via underlying miR-29-OXPHOS complex pathways. Furthermore, we infer reduced miR-29 expression compensatorily enhances insulin secretion under glucotoxicity.

Excess glucose alone depress young mesenchymal stromal/stem cell osteogenesis and mitochondria activity within hours/days via NAD+/SIRT1 axis

BackgroundThe impact of global overconsumption of simple sugars on bone health, which peaks in adolescence/early adulthood and correlates with osteoporosis (OP) and fracture risk decades, is unclear. Mesenchymal stromal/stem cells (MSCs) are the progenitors of osteoblasts/bone-forming cells, and known to decrease their osteogenic differentiation capacity with age. Alarmingly, while there is correlative evidence that adolescents consuming greatest amounts of simple sugars have the lowest bone mass, there is no mechanistic understanding on the causality of this correlation.MethodsBioinformatics analyses for energetics pathways involved during MSC differentiation using human cell information was performed. In vitro dissection of normal versus high glucose (HG) conditions on osteo-/adipo-lineage commitment and mitochondrial function was assessed using multi-sources of non-senescent human and murine MSCs; for in vivo validation, young mice was fed normal or HG-added water with subsequent analyses of bone marrow CD45− MSCs.ResultsBioinformatics analyses revealed mitochondrial and glucose-related metabolic pathways as integral to MSC osteo-/adipo-lineage commitment. Functionally, in vitro HG alone without differentiation induction decreased both MSC mitochondrial activity and osteogenesis while enhancing adipogenesis by 8 h’ time due to depletion of nicotinamide adenine dinucleotide (NAD+), a vital mitochondrial co-enzyme and co-factor to Sirtuin (SIRT) 1, a longevity gene also involved in osteogenesis. In vivo, HG intake in young mice depleted MSC NAD+, with oral NAD+ precursor supplementation rapidly reversing both mitochondrial decline and osteo-/adipo-commitment in a SIRT1-dependent fashion within 1 ~ 5 days.ConclusionsWe found a surprisingly rapid impact of excessive glucose, a single dietary factor, on MSC SIRT1 function and osteogenesis in youthful settings, and the crucial role of NAD+—a single molecule—on both MSC mitochondrial function and lineage commitment. These findings have strong implications on future global OP and disability risks in light of current worldwide overconsumption of simple sugars.

Mitochondrial calcium overload contributes to cannabinoid‐induced paraptosis in hormone‐responsive breast cancer cells

AbstractStudies have shown that natural products can induce paraptosis in tumour cell lines. Paraptosis is characterized by cytoplasmic vacuolation arising from the endoplasmic reticulum (ER) and mitochondria. The mechanism of paraptosis is unclear; however, dysregulation of Ca2+ homeostasis is believed to affect paraptosis induction. This study investigated the mechanism of cell death induced by a phytocannabinoid ratio in the MCF7 breast cancer cell line. The crystal violet assay was used to detect changes in viability and morphology changes were investigated using light and transmission electron microscopy. Various inhibitors, fluorescent staining with high‐content screening, and Western blot analysis were used to investigate different cell death mechanisms. The phytocannabinoid ratio induced significant cell death and cytoplasmic vacuolation in MCF7 cells; however, no apoptosis, necrosis, autophagy, or ferroptosis was detected. Vacuolation induced by phytocannabinoid treatment was inhibited by cycloheximide, suggesting paraptosis induction. The mechanism of paraptosis induction was investigated, and it was found that treatment (1) induced ER dilation and mitochondrial swelling, (2) induced significant ER stress and mitochondrial Ca2+ overload and dysfunction, which appeared to be mediated by the voltage‐dependent anion channel, and (3) significantly impaired all mitochondrial metabolic pathways. The data demonstrated that paraptosis induced by the cannabinoid ratio was mediated by Ca2+ flux from the ER to the mitochondria. These findings highlight a novel mechanism of cannabinoid‐induced cell death and emphasize the anti‐cancer potential of cannabinoid ratios, which exhibited enhanced effects compared to individual cannabinoids.

Abstract PO3-06-08: Epithelial to Mesenchymal Transition Confers Sensitivity to Cytotoxic Agent Ophiobolin A via Alterations in Mitochondrial Function and Metabolic Pathways

Abstract Epithelial to Mesenchymal Transition Confers Sensitivity to Cytotoxic Agent Ophiobolin A via Alterations in Mitochondrial Function and Metabolic Pathways Haleigh Parker1, Kayla Haberman1, Alexander Kornienko2, Antonio Evidente3, Daniel Romo4, Benny Abraham Kaipparettu5,6, Joseph Taube1,6 1 Department of Biology, Baylor University, Waco, TX, USA 2 Department of Chemistry and Biochemistry, Texas State University, San Marcos, TX, USA 3 Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario Monte Sant’Angelo, Naples, Italy 4 Department of Chemistry and Biochemistry, Baylor University, Waco, TX, USA 5 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA 6 Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX, USA Background Metastatic progression in patients with triple negative breast cancer (TNBC) occurs in approximately half of all patients, reducing median overall survival. Metastasis may be facilitated through the epithelial to mesenchymal transition (EMT), which generates cancer cells with enhanced metastatic capacity and resistance to chemotherapeutics, features which are partially mediated by alterations in metabolic pathways and mitochondrial function. Here, we show a drug-like small molecule possesses EMT-specific cytotoxic activity via EMT-imparted variations in metabolic and mitochondrial functions. The fungus-derived sesterterpenoid, Ophiobolin A (OpA), possesses nanomolar cytotoxic activity and a high therapeutic index, though its target and mechanism of action remain unknown. Objectives The objectives of this study are to characterize alterations in metabolic pathways and mitochondrial function in the context of EMT, determine OpA induced alterations in specific metabolic pathways involved in metastasis and identify metabolic targets for the treatment of chemotherapeutic-resistant, metastatic triple negative breast cancer. Methods This study identifies distinct, novel metabolic alterations associated with EMT status and specific changes in mitochondrial and metabolic function associated with OpA treatment via analysis of intermediate metabolites, rates of oxidative phosphorylation and glycolysis, metabolic pathway capacity and dependency, protein expression, iron localization, reactive oxygen species accumulation and transmission electron microscopy. Results Our analysis indicates that OpA acts in a mitochondria-specific manner to cause a loss of membrane potential in EMT-positive, but not EMT-negative, cells with specific effects on complex III of the electron transport chain and oxidative phosphorylation as a whole. Furthermore, distinct shifts in glycolysis not only support previous data, but also provides insight as to mitochondrial functionality. Cells were also found to have distinct metabolic pathway capacities/dependencies and ROS and iron accumulation, depending on EMT status and OpA treatment. Finally, specific morphological alterations were uncovered via TEM image analysis. Therefore, we conclude that EMT imparts alterations in mitochondrial function and metabolic pathways, conferring sensitivity to the cytotoxic effects of OpA. Citation Format: Haleigh Parker, Kayla Haberman, Joseph Taube, Alexander Kornienko, Antonio Evidente, Daniel Romo, Benny Kaipparettu. Epithelial to Mesenchymal Transition Confers Sensitivity to Cytotoxic Agent Ophiobolin A via Alterations in Mitochondrial Function and Metabolic Pathways [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-06-08.

Sex-Specific Mitochondrial Bioenergetics Differences in Heart and Kidney Cortex

Rationale: Mitochondrial bioenergetics of different tissues have been studied widely, but their sex-specific differences have not been systematically studied. The goal of this study was therefore to investigate the sex-specific differences in mitochondrial respiration (oxygen consumption rate; OCR) and membrane potential (Δψ) in the heart and kidney cortex and to explore underlying mechanisms responsible for any observed differences. Methods: Mitochondria were isolated from adult male and female Sprague-Dawley rat heart and kidney cortex and were used to study their bioenergetics responses. Three different substrate combinations, namely, pyruvate+malate (PM), glutamate+malate (GM), and succinate in the presence of complex I inhibitor rotenone (SR) were used. This was followed by two different ADP addition protocols to determine how different substrates influence mitochondrial OCR and Δψ to ADP perturbations during oxidative phosphorylation (OxPhos) in male and female heart and kidney cortex. In one, a single saturated dose of ADP, while in the other, sequentially increasing doses of ADP were added to mitochondria in the presence of different substrates. Mitochondrial OCR was measured using a dual chamber Oroboros Oxygraph-2k Instrument and Δψ was measured using a spectrofluorometer and rhodamine-123 dye. From these measurements, OCR and Δψ alterations under different respiratory states and as functions of added ADP concentration were determined for different substrates and for both male and female rats. Results: Male and female mitochondria exhibited distinct respiratory patterns in the kidney cortex and heart with different substrates. In the kidney cortex, male mitochondria showed significantly higher OCR than female mitochondria when fueled with PM or SR. However, no significant sex differences in the OCR were observed when GM were used as substrates. In contrast, heart mitochondria exhibited negligible sex differences in the OCR for PM and SR, but significant differences emerged with GM, where female mitochondria displayed higher OCR than their male counterparts. Interestingly, sex-specific variations of Δψ in ADP-stimulated respiration were not apparent for either tissue or substrate when a single saturated dose of ADP was used. Instead, a trend similar to the OCR emerged when ADP was administered in gradually increasing concentrations. Conclusion: Heart and kidney cortex mitochondrial Δψ for males and females did not differ conclusively with the two ADP addition protocols. However, there were significant differences in the OCR of mitochondria from the heart and kidney cortex of males and females, depending on the respiratory substrate utilized. Males were found to have enhanced OCR in the kidney cortex, whereas females exhibited higher OCR in the heart. These observed sex-specific differences in the OCR could be attributed to differential regulation of mitochondrial metabolic pathways by respiratory substrates and sex hormones. NIH R01-HL151587. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A tipping point in dihydroxyacetone exposure: mitochondrial stress and metabolic reprogramming alter survival in rat cardiomyocytes H9c2 cells

Exogenous exposures to the triose sugar dihydroxyacetone (DHA) occur from sunless tanning products and electronic cigarette aerosol. Once inhaled or absorbed, DHA enters cells, is converted to dihydroxyacetone phosphate (DHAP), and incorporated into several metabolic pathways. Cytotoxic effects of DHA vary across the cell types depending on the metabolic needs of the cells, and differences in the generation of reactive oxygen species (ROS), cell cycle arrest, and mitochondrial dysfunction have been reported. We have shown that cytotoxic doses of DHA induced metabolic imbalances in glycolysis and oxidative phosphorylation in liver and kidney cell models. Here, we examine the dose-dependent effects of DHA on the rat cardiomyocyte cell line, H9c2. Cells begin to experience cytotoxic effects at low millimolar doses, but an increase in cell survival was observed at 2 mM DHA. We confirmed that 2 mM DHA increased cell survival compared to the low cytotoxic 1 mM dose and investigated the metabolic differences between these two low DHA doses. Exposure to 1 mM DHA showed changes in the cell's fuel utilization, mitochondrial reactive oxygen species (ROS), and transient changes in the glycolysis and mitochondrial energetics, which normalized 24 h after exposure. The 2 mM dose induced robust changes in mitochondrial flux through acetyl CoA and elevated expression of fatty acid synthase. Distinct from the 1 mM dose, the 2 mM exposure increased mitochondrial ROS and NAD(P)H levels, and sustained changes in LDHA/LDHB and acetyl CoA-associated enzymes were observed. Although the cells were exposed to low cytotoxic (1 mM) and non-cytotoxic (2 mM) acute doses of DHA, significant changes in mitochondrial metabolic pathways occurred. Further, the proliferation increase at the acute 2 mM DHA dose suggests a metabolic adaption occurred with sustained consequences in survival and proliferation. With increased exogenous exposure to DHA through e-cigarette aerosol, this work suggests cell metabolic changes induced by acute or potentially chronic exposures could impact cell function and survival.

Mitochondrial Proteins as Metabolic Biomarkers and Sites for Therapeutic Intervention in Primary and Metastatic Cancers

Abstract: Accelerated aerobic glycolysis is one of the main metabolic alterations in cancer, associated with malignancy and tumor growth. Although glycolysis is one of the most studied properties of tumor cells, recent studies demonstrate that oxidative phosphorylation (OxPhos) is the main ATP provider for the growth and development of cancer. In this last regard, the levels of mRNA and protein of OxPhos enzymes and transporters (including glutaminolysis, acetate and ketone bodies catabolism, free fatty acid β-oxidation, Krebs Cycle, respiratory chain, phosphorylating system- ATP synthase, ATP/ADP translocator, Pi carrier) are altered in tumors and cancer cells in comparison to healthy tissues and organs, and non-cancer cells. Both energy metabolism pathways are tightly regulated by transcriptional factors, oncogenes, and tumor-suppressor genes, all of which dictate their protein levels depending on the micro-environmental conditions and the type of cancer cell, favoring cancer cell adaptation and growth. In the present review paper, variation in the mRNA and protein levels as well as in the enzyme/ transporter activities of the OxPhos machinery is analyzed. An integral omics approach to mitochondrial energy metabolism pathways may allow for identifying their use as suitable, reliable biomarkers for early detection of cancer development and metastasis, and for envisioned novel, alternative therapies.

Metabolic Flexibility: Targeting Mitochondrial Dynamics in Cancer Therapy

Abstract Various types of cancer exhibit distinct sensitivities to anticancer drugs, reflecting their unique developmental pathways. Exploiting mitochondrial dysfunction in cancer cells presents novel therapeutic opportunities. In recent years, advancements have underscored the significance of oxidative phosphorylation, fatty acid oxidation, and glutamine addiction in cancer cells, shifting attention towards mitochondrial metabolism. The foundation of mitochondrial medicine lies in comprehending targeted mitochondrial therapy and the development of specific mitochondrial drugs. Certain alterations within mitochondrial metabolism lead to the accumulation of oncometabolites, serving as epigenetic regulators, or an increase in reactive oxygen species production, both of which contribute to tumorigenesis. Importantly, the substantial involvement of mitochondrial metabolism in tumorigenesis offers potential avenues for exploiting as strategies for cancer therapy. Integrated pharmacological approaches targeting mitochondrial metabolic pathways are imperative for an effective eradication of tumor masses and a concurrent elimination of small subpopulations of mitochondria. The significance of mitochondria in cancer is undeniable, offering numerous appealing targets for both tailored and personalized cancer therapy.

Abstract 3331: Preclinical combination of ONC206 with radiotherapy and temozolomide in a GBM mouse orthotopic model

Abstract Glioblastoma multiforme (GBM) is the most common form of primary malignant brain tumor in adults. It is also the most aggressive and lethal. Standard of care therapy comprises maximal safe surgical resection followed by adjuvant alkylating agent temozolomide (TMZ) and radiotherapy (RT). There have only been five drugs and one device ever approved by the FDA for the treatment of GBM. The five-year survival rate for GBM patients has shown no notable improvement in the last three decades. We previously reported that the first-in-class imipridone small-molecule Dordaviprone (ONC201) decreases protein chaperone ClpX to unleash mitochondrial protease ClpP activity, integrated stress response and cell death. Preclinical combination of ONC201 (100 mg/kg weekly) with radiotherapy and Temozolomide in a GBM mouse orthotopic model results in reduced tumor burden and prolonged survival. The second generation imipridone ONC206 also activates integrated stress response and decreases ClpX in GBM cells but ONC206 is approximately 20 times more potent than its parent imipridone ONC201. ONC206 0.08 uM in combination with 12.5 uM TMZ and 2 Gy RT reduced the cell viability of GBM cells significantly compared to all single treatments and double treatments. During a short-term treatment of a GBM mouse orthotopic model, ONC206 (50 mg/kg weekly, half of ONC201 doses) synergizes with TMZ and RT to induce more tumor cell apoptosis and inhibits more tumor cell proliferation compared to other groups with single or double treatments. There are two ongoing clinical trials with ONC206: single weekly or multiple-day weekly dose regimens of single-agent, oral ONC206 in patients with recurrent, primary central nervous system (CNS) neoplasms (NCT04541082) and ONC206 alone or in combination with radiation therapy in treating patients with diffuse midline gliomas that is newly diagnosed or has come back (recurrent) or other recurrent primary malignant CNS tumors (NCT04732065). Future studies evaluating the role of immune function, mitochondrial metabolism, dopamine receptors, the ISR and TRAIL pathway in the synergistic effect would support further development of the triple combination regimen of ONC206, TMZ and radiation therapy for GBM clinical trial. Citation Format: Lanlan Zhou, Leiqing Zhang, Jun Zhang, Shengliang Zhang, Wafik S. El-Deiry. Preclinical combination of ONC206 with radiotherapy and temozolomide in a GBM mouse orthotopic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3331.

Abstract 7429: A web-based application to co-register elemental imaging with histopathology to enhance the study of metal bioaccumulation within tumors

Abstract Essential trace elements are crucial in various biological processes, including tumor growth, migration, and metastasis. For instance, copper ions are involved in mitochondrial metabolism, cell proliferation, tumor migration, metastasis and proangiongenic pathways. Localizing where metal ion homeostasis is disrupted is vital for understanding these processes beyond what bulk analysis can reveal. Elemental imaging offers high-resolution, quantifiable multi-elemental distribution maps, providing a more detailed analysis compared to traditional bulk measurements. However, aligning findings with tissue structures identified in histopathological data can be challenging. To address this, we developed a web-based application for the co-registration and analysis of Whole Slide Images (WSI) and elemental imaging data at the cellular level. The application integrates WSI with elemental maps generated by technologies such as ultrafast laser ablation and inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOF-MS), as well as a range of other elemental imaging techniques. Hosted by the Biomedical National Elemental Imaging Resource, the current application allows for a streamlined data integration, co-registration, and analysis, including: 1) uploading WSI, elemental maps, and pathologist annotations, 2) automated preprocessing to identify tissue regions, 3) co-registration to merge WSI and elemental maps, 4) custom import, real-time overlay/editing of pathologist annotations transferred from WSI, 5) plotting, statistical tools and 6) data export. This application enables users to compare elemental abundance within/between tissue structures annotated on H&E or immunohistochemical slides. Derived data can pinpoint elements and mixtures whose abundance differs significantly across various pathological conditions. As an initial test, we applied the software on breast HER2/ER/PR tumors, varying in molecular subtype/histology. Preliminary results, using Bayesian lognormal regression models, adjusting for tumor subtypes, indicated a subtype-dependent variation in copper abundance. The relative difference in copper levels between tumor and stromal regions was notably less in HER2 tumors (p=0.026) compared to HR tumors (p<0.0001). Conversely, iron showed a consistently higher abundance in stromal areas across both subtypes (p<0.0001). Future enhancements include improved co-registration capabilities, hotspot analysis, machine learning, mixture effects, and cloud-based architecture. This method uniquely enables integration of elemental data with spatial transcriptomics, mapping gene transcripts on tissue slides. With current ST assays aligned with H&E slides, this tool facilitates co-registration, offering insights into biological pathways affected by metal homeostasis disruptions. Citation Format: Yunrui Lu, Ramsey Steiner, Serin Han, Matthew Chan, Tracy Punshon, Brian Jackson, Linda Vahdat, Jonathan Marotti, Joshua Levy. A web-based application to co-register elemental imaging with histopathology to enhance the study of metal bioaccumulation within tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7429.

Abstract 2019: Primary cancer associated fibroblasts increase the chemoresistance of triple negative breast cancer cells by mitochondria metabolic upregulation

Abstract Breast cancers (BCs) are the most prevalent cancers occurring in women. Tumor reoccurrence attributed to chemotherapy resistance remains a main challenge in fighting disease relapse. Cancer associated fibroblasts (CAFs) are the most prominent component of the tumor microenvironment (TME) and have already been implicated in therapy resistances. Although the exact molecular mechanisms behind this resistance remain unclear. In this regard, we recently identified 4 CAF populations (S1-S4) in ovarian and BCs, and showed that only CAF-S1 promote immunotherapy resistance as well as metastasis. Based on these observations, we are now investigating the impact of CAF-S1 on the chemotherapy response of BCs. To study the interactions between CAF-S1 and BC cells, we developed a tumor on chip (ToC) device, mimicking the TME in 3D. Using ToC live imaging, we show that primary CAF-S1 mediate a chemoprotective effect over luminal and triple negative BC (TNBC) cells by significantly decreasing their apoptosis rate as early as 10h after Doxorubicin and 40h after Paclitaxel treatment. Interestingly, CAF-S1 display a highly heterogenous response to Doxorubicin, allowing the classification into chemo-sensitive and -resistant CAF-S1. Regardless of premature CAF-S1 cell death, the chemoprotective effect over BC cells is maintained for at least 3 days. This indicates a crucial CAF-S1 - cancer cell cross talk at early timepoints. Additionally, by applying machine learning algorithms on live imaging videos, we demonstrate that physical proximity of CAF-S1 to BC cells immediately after treatment initiation correlates with BC cell survival, even after several days of treatment. This again implies cancer cell priming by CAF-S1 as a possible mechanism of chemotherapy resistance. To investigate this molecular interplay further, we performed scRNA-seq of ToC extracted and treatment naïve CAF-S1 and TNBC cells as well as bulk RNA-seq of patient derived TNBC tumors before/after chemotherapy treatment. We identify mitochondrial metabolic pathways to be upregulated in TNBC cells after CAF-S1 co-culture inside ToC devices and in TNBC cells of chemo-resistant patients enriched in CAF-S1 at baseline. We are now validating the impact of target gene knock downs on the survival of TNBC cells in CAF-S1 co-cultures after chemotherapy treatment on chip. Overall, these results provide insights into molecular mechanisms by which CAF-S1 promote chemotherapy resistance of breast cancers. Citation Format: Isabella Hofer, Arianna Mencattini, Yann Kieffer, Eugenio Martinelli, Maria Carla Parrini, Fatima Mechta-Grigoriou. Primary cancer associated fibroblasts increase the chemoresistance of triple negative breast cancer cells by mitochondria metabolic upregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2019.

CYP14 family in Caenorhabditis elegans: Mitochondrial function, detoxification, and lifespan.

CYP-14 members of the Caenorhabditis elegans (C. elegans) Cytochrome P450 (CYP) enzyme family, plays important roles in mitochondrial dysfunction, detoxification, lipid metabolism, defense and lifespan regulation. The review identifies CYP-14 members: cyp-14A1, cyp-14A2, cyp-14A3, cyp-14A4, cyp-14A5 and their homology with human CYP families. Despite limited studies on C. elegans cyp-14 members, the findings unraveled their complex crosstalk between mitochondrial stress, detoxification mechanisms, and lifespan regulation, emphasizing the complexity of these interconnected pathways as well as how their regulation depends on environmental cues changes including pH, nutrients, ROS and chemical stressors. The review underscores the translational relevance to human health, shedding light on potential human homologues and their implications in age-related, metabolic and respiratory diseases. Among other genes, cyp-14A2 and cyp-14A4 predominate the mitochondrial function, heat resistance, lipid metabolism, detoxification and lifespan pathways. In conclusion, these insights pave the way for future research, offering promising avenues for therapeutic interventions targeting CYP-14 activity to address age-related diseases and promote healthy aging.

Divergent mitochondrial responses and metabolic signal pathways secure the azole resistance in Crabtree-positive and negative Candida species.

Azole drugs are the main therapeutic drugs for invasive fungal infections. However, azole-resistant strains appear repeatedly in the environment, posing a major threat to human health. Several reports have shown that mitochondria are associated with the virulence of pathogenic fungi. However, there are few studies on the mechanisms of mitochondria-mediated azoles resistance. Here, we first performed mitochondrial proteomic analysis on multiple Candida species (Candida albicans, Nakaseomyces glabrata, Pichia kudriavzevii, and Candida auris) and analyzed the differentially expressed mitochondrial proteins (DEMPs) between azole-sensitive and azole-resistant Candida species. Subsequently, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, gene ontology analysis, and protein-protein interaction network analysis of DEMPs. Our results showed that a total of 417, 165, and 25 DEMPs were identified in resistant C. albicans, N. glabrata, and C. auris, respectively. These DEMPs were enriched in ribosomal biogenesis at cytosol and mitochondria, tricarboxylic acid cycle, glycolysis, transporters, ergosterol, and cell wall mannan biosynthesis. The high activations of these cellular activities, found in C. albicans and C. auris (at low scale), were mostly opposite to those observed in two fermenter species-N. glabrata and P. kudriavzevii. Several transcription factors including Rtg3 were highly produced in resistant C. albicans that experienced a complex I activation of mitochondrial electron transport chain (ETC). The reduction of mitochondrial-related activities and complex IV/V of ETC in N. glabrata and P. kudriavzevii was companying with the reduced proteins of Tor1, Hog1, and Snf1/Snf4.IMPORTANCECandida spp. are common organisms that cause a variety of invasive diseases. However, Candida spp. are resistant to azoles, which hinders antifungal therapy. Exploring the drug-resistance mechanism of pathogenic Candida spp. will help improve the prevention and control strategy and discover new targets. Mitochondria, as an important organelle in eukaryotic cells, are closely related to a variety of cellular activities. However, the role of mitochondrial proteins in mediating azole resistance in Candida spp. has not been elucidated. Here, we analyzed the mitochondrial proteins and signaling pathways that mediate azole resistance in Candida spp. to provide ideas and references for solving the problem of azole resistance. Our work may offer new insights into the connection between mitochondria and azoles resistance in pathogenic fungi and highlight the potential clinical value of mitochondrial proteins in the treatment of invasive fungal infections.

GLP-1 receptor agonist attenuates tubular cell ferroptosis in diabetes via enhancing AMPK-fatty acid metabolism pathway through macropinocytosis

Kidney tubules are mostly responsible for pathogenesis of diabetic kidney disease. Actively reabsorption of iron, high rate of lipid metabolism and exposure to concentrated redox-active compounds constructed the three main pillars of ferroptosis in tubular cells. However, limited evidence has indicated that ferroptosis is indispensable for diabetic tubular injury. Glucagon-like peptide-1 receptor agonist (GLP-1RA) processed strong benefits on kidney outcomes in people with diabetes. Moreover, GLP-1RA may have additive effects by improving dysmetabolism besides glucose control and weight loss. Therefore, the present study aimed at exploring the benefits of exendin-4, a high affinity GLP-1RA on kidney tubular dysregulation in diabetes and the possible mechanisms involved, with focus on ferroptosis and adenosine 5′-monophosphate-activated protein kinase (AMPK)-mitochondrial lipid metabolism pathway. Our data revealed that exendin-4 treatment markedly improved kidney structure and function by reducing iron overload, oxidative stress, and ACSL4-driven lipid peroxidation taken place in diabetic kidney tubules, along with reduced GPX4 expression and GSH content. AMPK signaling was identified as the downstream target of exendin-4, and enhancement of AMPK triggered the transmit of its downstream signal to activate fatty acid oxidation in mitochondria and suppress lipid synthesis and glycolysis, and ultimately alleviated toxic lipid accumulation and ferroptosis. Further study suggested that exendin-4 was taken up by tubular cells via macropinocytosis. The protective effect of exendin-4 on tubular ferroptosis was abolished by macropinocytosis blockade. Taken together, present work demonstrated the beneficial effects of GLP-1RA treatment on kidney tubular protection in diabetes by suppressing ferroptosis through enhancing AMPK-fatty acid metabolic signaling via macropinocytosis.

Abstract A049: Epithelial to mesenchymal transition confers sensitivity to cytotoxic agent Ophiobolin A via alterations in mitochondrial function and metabolic pathways

Abstract Triple negative breast cancer (TNBC) is characterized by its high metastatic potential, resistance to therapeutics and elevated degree of disease reoccurrence. In patients with TNBC, metastasis occurs in approximately half of all cases and is tightly correlated with poor overall survival, made worse by limited therapeutic options. Metastatic events are hypothesized to be facilitated by the epithelial to mesenchymal transition (EMT), gaining enhanced self-renewal, migration, invasion and multidrug resistant capabilities. Thus, targeting these cells remains a primary therapeutic goal, despite significant difficulty. Previous attempts in pursuing these cells through cytotoxic chemotherapeutics or targeted agents has largely failed to significantly improve patient outcomes. However, the fungus-derived sesterterpenoid Ophiobolin A (OpA) has demonstrated nanomolar cytotoxic capabilities and a high therapeutic index, specific to EMT+cells in breast cancer. The properties conferring this sensitivity, and possible novel therapeutic target, though, remain unclear. As the drug-like small molecule presents a major therapeutic opportunity, the details of its mechanism of action must be determined. To this end, we generated mitochondrial cybrids and quantified activity in individual electron transport chain (ETC) complexes, and found OpA to act in a mitochondria-targeted manner. Expanding upon this, we designed an EMT based model of breast cancer and examined alterations in mitochondrial function and metabolic pathways. Targeted liquid chromatography-mass spectronomy of metabolites indicated significant variations in many of those involved in glycolysis, OXPHOS and glutaminolysis. Flow cytometry further revealed a significant decrease in membrane potential and increase in mitochondrial reactive oxygen species (ROS), indicating mitochondrial damage. Dynamic changes between OXPHOS and glycolysis were also detected via Seahorse Bioanalyzer. Transmission electron microscopy (TEM) images and fluorescent microscopy showed distinct alterations in mitochondrial morphology, spatial dynamics and network integrity. Finally, using targeted fluorescent staining, an increase in mitochondrial labile iron was uncovered. This work reveals novel metabolic variations in the context of EMT and exploitable therapeutic targets. Thus, based on these EMT-specific responses, we conclude that EMT imparts distinct alterations to mitochondrial function and metabolic pathways, conferring sensitivity to the cytotoxic effects of OpA. Citation Format: Haleigh Parker, Kayla Haberman, Alexander Kornienko, Antonio Evidente, Daniel Romo, Benny Kaipparettu, Joseph Taube. Epithelial to mesenchymal transition confers sensitivity to cytotoxic agent Ophiobolin A via alterations in mitochondrial function and metabolic pathways [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A049.

Mitochondrial dynamics and metabolic regulation control T cell fate in the thymus.

Several studies demonstrated that mitochondrial dynamics and metabolic pathways control T cell fate in the periphery. However, little is known about their implication in thymocyte development. Our results showed that thymic progenitors (CD3-CD4-CD8- triple negative, TN), in active division, have essentially a fused mitochondrial morphology and rely on high glycolysis and mitochondrial oxidative phosphorylation (OXPHOS). As TN cells differentiate to double positive (DP, CD4+CD8+) and single positive (SP, CD4+ and CD8+) stages, they became more quiescent, their mitochondria fragment and they downregulate glycolysis and OXPHOS. Accordingly, in vitro inhibition of the mitochondrial fission during progenitor differentiation on OP9-DL4 stroma, affected the TN to DP thymocyte transition by enhancing the percentage of TN and reducing that of DP, leading to a decrease in the total number of thymic cells including SP T cells. We demonstrated that the stage 3 triple negative pre-T (TN3) and the stage 4 triple negative pre-T (TN4) have different metabolic and functional behaviors. While their mitochondrial morphologies are both essentially fused, the LC-MS based analysis of their metabolome showed that they are distinct: TN3 rely more on OXPHOS whereas TN4 are more glycolytic. In line with this, TN4 display an increased Hexokinase II expression in comparison to TN3, associated with high proliferation and glycolysis. The in vivo inhibition of glycolysis using 2-deoxyglucose (2-DG) and the absence of IL-7 signaling, led to a decline in glucose metabolism and mitochondrial membrane potential. In addition, the glucose/IL-7R connection affects the TN3 to TN4 transition (also called β-selection transition), by enhancing the percentage of TN3, leading to a decrease in the total number of thymocytes. Thus, we identified additional components, essential during β-selection transition and playing a major role in thymic development.

Amyloid beta 42 alters cardiac metabolism and impairs cardiac function in male mice with obesity

There are epidemiological associations between obesity and type 2 diabetes, cardiovascular disease and Alzheimer’s disease. The role of amyloid beta 42 (Aβ42) in these diverse chronic diseases is obscure. Here we show that adipose tissue releases Aβ42, which is increased from adipose tissue of male mice with obesity and is associated with higher plasma Aβ42. Increasing circulating Aβ42 levels in male mice without obesity has no effect on systemic glucose homeostasis but has obesity-like effects on the heart, including reduced cardiac glucose clearance and impaired cardiac function. The closely related Aβ40 isoform does not have these same effects on the heart. Administration of an Aβ-neutralising antibody prevents obesity-induced cardiac dysfunction and hypertrophy. Furthermore, Aβ-neutralising antibody administration in established obesity prevents further deterioration of cardiac function. Multi-contrast transcriptomic analyses reveal that Aβ42 impacts pathways of mitochondrial metabolism and exposure of cardiomyocytes to Aβ42 inhibits mitochondrial complex I. These data reveal a role for systemic Aβ42 in the development of cardiac disease in obesity and suggest that therapeutics designed for Alzheimer’s disease could be effective in combating obesity-induced heart failure.

Iguratimod Regulates CD4+ T-Cell Homeostasis and Function By Restoring PINK1/Parkin-Mediated Mitophagy in Immune Thrombocytopenia

Introduction Immune thrombocytopenia (ITP), an acquired autoimmune disease, is characterized by immune-mediated platelet destruction and suppressed platelet production, resulting in isolated thrombocytopenia. Subpopulation imbalance and dysfunction of CD4+ T cells are involved in the pathogenesis of ITP. However, the exact mechanism of CD4+ T-cell abnormalities is not fully understood. Mitophagy, a process that eliminates damaged and dysfunctional mitochondria, has conventionally been considered the primary mechanism responsible for mitochondrial quality control. Mitophagy deficiency has been implicated in a series of inflammatory and autoimmune diseases, such as systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD) and rheumatoid arthritis (RA). However, whether it plays a role in ITP immune abnormalities and its specific mechanism remain unclear. Iguratimod is a small molecule compound that is widely used as a novel antirheumatic drug in the treatment of rheumatoid arthritis. Iguratimod inhibits the production of several inflammatory cytokines, including IL-1, IL-6, IL-8, and tumor necrosis factor (TNF), by enhancing mitophagy. Our previous clinical study demonstrated the efficacy of iguratimod in the treatment of ITP. This study aimed to explore whether iguratimod regulates CD4+ T-cell homeostasis and function by enhancing mitophagy in ITP. Methods BM CD4+ T cells were isolated from ITP patients and healthy donors. The subgroup of CD4+ T cells was analyzed by flow cytometry. ELISA was used to evaluate cytokine and chemokine levels in bone marrow plasma. Mitochondrial mass, morphology, quality and mitophagy were evaluated by flow cytometry, confocal microscopy, electron microscopy and western blotting. RNA sequencing and metabolomics were performed. Epigenetic state alteration was assessed by a transposase-accessible chromatin assay with high throughput sequencing (ATAC-seq). Iguratimod-treated BM-derived ITP-CD4+ T cells and an ITP active mouse model were used to explore its regulation of mitophagy and effects on T-cell homeostasis and function. Results Th1/Th2 imbalance and Treg reduction were observed in the bone marrow of ITP patients, consistent with a previous report. Flow cytometry and confocal microscopy revealed an increase in mitochondrial content and mtROS in CD4+ T cells but a decrease in mitochondrial membrane potential. Furthermore, electron microscopy revealed structural damage to mitochondria in ITP-CD4+ T cells. These data confirmed an accumulation of damaged mitochondria in ITP-CD4+ T cells. The expression levels of LC3II/LC3I, PINK1 and parkin decreased in CD4+ T cells, and the colocalization of mitochondria and lysosomes decreased, which suggested compromised PINK1/parkin-mediated mitophagy in ITP-CD4+ T cells. RNA-seq revealed transcriptional changes associated with mitochondrial function, mitophagy and metabolic pathways in ITP-CD4+ T cells. Moreover, metabolomics showed alterations in TCA cycle metabolites. Since epigenetic programs are regulated by metabolites, ATAC-seq was performed to obtain the epigenetic state. We found that ITP-CD4+ T cells altered chromatin accessibility in regions with T cell differentiation, transcription factors and T-cell function. After in vitro treatment with iguratimod, the Th1/Th2 imbalance was corrected, and the number of Tregs was restored. The content, morphology and membrane potential of mitochondria in ITP-CD4+ T cells were restored after iguratimod intervention. The expression level of PINK1 and parkin and the colocalization of mitochondria and lysosomes were increased, suggesting an improvement in mitophagy. In vivo, iguratimod accelerated platelet recovery in an ITP active mouse model. After treatment, the Th1/Th2 imbalance and the Treg number were restored, and the mitophagy of CD4+ T cells was corrected in the bone marrow of the ITP mouse model. Conclusion PINK1/Parkin-mediated mitophagy deficiency contributed to mitochondrial dysfunction and altered the metabolism of ITP-CD4+ T cells, which affected their differentiation and function by regulating the epigenetic state of immune response genes. Iguratimod could restore CD4+ T-cell subpopulations and treat ITP by enhancing mitophagy.

Analysis of Metabolic Mechanism of CD19CAR-T with CD79A/CD40 Co-Stimulatory Domain Based on Comparison with Established CAR-Ts

Introduction: Anti-CD19 chimeric antigen receptor T cell (CD19CAR-T) therapy has achieved significant clinical outcomes and changed treatment strategy in B-cell malignancies. However, recurrence after CAR-T therapy remains a major obstacle due to poor peak expansion and short persistence of CAR-T cells. We have reported that CD19CAR-T incorporating a co-stimulatory domain consisting of CD79A and CD40 (79A40CAR) provided superior anti-tumor efficacy and survival compared to CD19CAR-T with CD28 or 4-1BB (CD28CAR/4-1BBCAR) in mice xenograft models. 79A40CAR exhibited sustained NF-κB phosphorylation and superior in vivo efficacy compared to the CD28CAR or 4-1BBCAR. (Molecular Therapy 2021, Julamanee J et al.) In this study, we aimed to clarify the molecular mechanism of 79A40CAR by comparing with CD28/4-1BBCAR in terms of T-cell metabolism. Methods: CD8+T cells were isolated from PBMCs of healthy donors and retrovirally transduced with either CD28IC-, 4-1BBIC-, or 79A40IC-CD19CARs. CD28CAR, 4-1BBCAR, or 79A40CAR were cocultured with 100 Gy irradiated NALM6 at E:T ratio = 1:5, respectively. Repeated antigen stimulations (Ag-stim) were performed, and day 7 after each stimulation was subjected to analysis. RNA-seq, expansion capacity, T-cell phenotype, and metabolic evaluation using a seahorse flux analyzer (Agilent Technologies) were performed. After Ag-stim, CD45RA negative CD62L positive and CD45RA negative CD62L positive CAR-T cell fractions were sorted as central memory (T CM) and effector memory (T EM), respectively. T CM and T EM received Ag-stim. After Ag-stim, we performed evaluation of mitochondrial functionality with MitoTracker based on mitochondrial mass and membrane potential. Results: All CD19CAR genes were successfully transduced into primary CD8+ T-cells. To understand the differences between co-stimulatory domains, we first confirmed bulk RNA-seq, expansion capacity, and T-cell phenotype. After Ag-stim, 79A40CAR showed enhancement of the NF-κB pathway. We detected few DEGs when 79A40CAR compared to 4-1BBCAR, however, we detected 162 upregulated DEGs when 79A40CAR compared to CD28CAR. Principal component analysis of gene expression demonstrated similarity between 4-1BBCAR and 79A40CAR. We confirmed that 79A40CAR had superior expansion capacity than CD28, or 4-1BBCAR after repeated Ag-stim. PD-1 and Tim-3 expression in 79A40CAR was lowest after repeated Ag-stim. In the evaluation of T-cell differentiation, 4-1BB and 79A40CAR showed an earlier transition to the effector phenotype compared to CD28CAR after Ag-stim. To evaluate differences in terms of T-cell metabolism, we performed metabolic analysis using a seahorse flux analyzer. After repeated Ag-stim, 4-1BB and 79A40CAR showed a predominant oxidative phosphorylation metabolic pathway (Fig1A) and higher spare respiratory capacity (SRC), which has been correlated with mitochondrial functionality (Fig1B), than CD28CAR. After repeated Ag-stim, 4-1BB and 79A40CAR demonstrated higher ECAR than CD28CAR. Because we observed differences in the expression of T-cell differentiation phenotype, we performed cell sorting to evaluate T-cell metabolism of each differentiation phenotype. After applying repeated Ag-stim to T CM and T EM-derived CD19CAR-T, mitochondrial functionality was evaluated using Mitotracker. T CM- (Fig1C) and T EM- (Fig1D) derived 4-1BB and 79A40CAR had more functional mitochondria and fewer dysfunctional mitochondria compared to CD28CAR. Conclusion: 79A40CAR seems to preserve the mitochondrial oxidative phosphorylation-dominant metabolic pathway and supply energy demands for a longer period, especially when compared to CD28CAR. 79A40CAR showed functionally very similar to 4-1BBCAR. We showed that CD19CAR-Ts had different metabolic pathway according to the different co-stimulatory domains. To reveal additional metabolic pathway differences between 4-1BBCAR and 79A40CAR, we are now conducting proteomics analysis.