bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2023‒07‒02
35 papers selected by
Kelsey Fisher-Wellman
East Carolina University

  1. Mol Cancer Res. 2023 Jun 26. pii: MCR-23-0049. [Epub ahead of print]
      When the electron transport chain (ETC) function is impaired, cancer cells rely on reductive carboxylation (RC) to convert α-ketoglutarate (αKG) to citrate for macromolecular synthesis, thereby promoting tumor growth. Currently, there is no viable therapy to inhibit RC for cancer treatment. In this study, we demonstrate that the mitochondrial uncoupler treatment effectively inhibits RC in cancer cells. Mitochondrial uncoupler treatment activates the ETC and increases the NAD+/NADH ratio. Using U-13C-glutamine and 1-13C-glutamine tracers, we show that mitochondrial uncoupling accelerates the oxidative TCA cycle and blocks RC under hypoxia, in von Hippel-Lindau (VHL) tumor suppressor-deficient kidney cancer cells, or under anchorage-independent growth condition. Together, these data demonstrate that mitochondrial uncoupling redirects α-KG from RC back to the oxidative TCA cycle, highlighting that the NAD+/NADH ratio is one key switch that determines the metabolic fate of α-KG. Inhibiting RC could be a key mechanism by which mitochondrial uncouplers inhibit tumor growth.
  2. Biomedicines. 2023 May 31. pii: 1598. [Epub ahead of print]11(6):
      Transcription of the mitochondrial genome is essential for the maintenance of oxidative phosphorylation (OXPHOS) and other functions directly related to this unique genome. Considerable evidence suggests that mitochondrial transcription is dysregulated in cancer and cancer metastasis and contributes significantly to cancer cell metabolism. Recently, inhibitors of the mitochondrial DNA-dependent RNA polymerase (POLRMT) were identified as potentially attractive new anti-cancer compounds. These molecules (IMT1, IMT1B) inactivate cancer cell metabolism through reduced transcription of mitochondrially-encoded OXPHOS subunits such as ND1-5 (Complex I) and COI-IV (Complex IV). Studies from our lab have discovered small molecule regulators of the mitochondrial matrix caseinolytic protease (ClpP) as probable inhibitors of mitochondrial transcription. These compounds activate ClpP proteolysis and lead to the rapid depletion of POLRMT and other matrix proteins, resulting in inhibition of mitochondrial transcription and growth arrest. Herein we present a comparison of POLRMT inhibition and ClpP activation, both conceptually and experimentally, and evaluate the results of these treatments on mitochondrial transcription, inhibition of OXPHOS, and ultimately cancer cell growth. We discuss the potential for targeting mitochondrial transcription as a cancer cell vulnerability.
    Keywords:  ClpP; POLRMT; mechanism of action; mitochondria; mitochondrial DNA; mitochondrial transcription; oxidative phosphorylation
  3. Cell Death Discov. 2023 Jun 29. 9(1): 203
      Cancer cells often hijack metabolic pathways to obtain the energy required to sustain their proliferation. Understanding the molecular mechanisms underlying cancer cell metabolism is key to fine-tune the metabolic preference of specific tumors, and potentially offer new therapeutic strategies. Here, we show that the pharmacological inhibition of mitochondrial Complex V delays the cell cycle by arresting breast cancer cell models in the G0/G1 phase. Under these conditions, the abundance of the multifunctional protein Aurora kinase A/AURKA is specifically lowered. We then demonstrate that AURKA functionally interacts with the mitochondrial Complex V core subunits ATP5F1A and ATP5F1B. Altering the AURKA/ATP5F1A/ATP5F1B nexus is sufficient to trigger G0/G1 arrest, and this is accompanied by decreased glycolysis and mitochondrial respiration rates. Last, we discover that the roles of the AURKA/ATP5F1A/ATP5F1B nexus depend on the specific metabolic propensity of triple-negative breast cancer cell lines, where they correlate with cell fate. On one hand, the nexus induces G0/G1 arrest in cells relying on oxidative phosphorylation as the main source of energy. On the other hand, it allows to bypass cell cycle arrest and it triggers cell death in cells with a glycolytic metabolism. Altogether, we provide evidence that AURKA and mitochondrial Complex V subunits cooperate to maintain cell metabolism in breast cancer cells. Our work paves the way to novel anti-cancer therapies targeting the AURKA/ATP5F1A/ATP5F1B nexus to lower cancer cell metabolism and proliferation.
  4. J Cell Sci. 2023 Jun 26. pii: jcs.260419. [Epub ahead of print]
      Cancer associated fibroblasts (CAFs) have distinct roles within the tumor microenvironment, which may impact the mode and efficacy of tumor cell migration. CAFs are known to increase invasion of less-aggressive breast cancer cells through matrix remodeling and leader-follower dynamics. Here, we demonstrate that CAFs communicate with breast cancer cells through the formation of contact-dependent tunneling nanotubes (TNTs) that allow for the exchange of cargo between cell types. The transferring of CAF mitochondria is an integral cargo component, and CAF mitochondria are sufficient to increase the 3D migration of cancer cells. This cargo transfer results in an increase in mitochondrial ATP production in cancer cells while having negligible impact on glycolytic ATP production. Manually increasing mitochondrial oxidative phosphorylation (OXPHOS) by providing extra substrates for OXPHOS fails to enhance cancer cell migration unless glycolysis is maintained at a constant level. Together, these data indicate that tumor-stromal crosstalk via TNTs and the associated metabolic symbiosis is a finely controlled mechanism by which tumor cells co-opt their microenvironment to promote cancer progression and may become a potential therapeutic target.
    Keywords:  ATP production.; Bioenergetics; CAF; Reverse Warburg effect; Tumor microenvironment; Tumor spheroid; Tunneling nanotube
  5. Cancer Res Commun. 2023 Apr;3(4): 709-724
      Most cancer-related deaths are due to metastases. Systemic factors, such as lipid-enriched environments [as low-density lipoprotein (LDL)-cholesterol], favor breast cancer, including triple-negative breast cancer (TNBC) metastasis formation. Mitochondria metabolism impacts TNBC invasive behavior but its involvement in a lipid-enriched setting is undisclosed. Here we show that LDL increases lipid droplets, induces CD36 and augments TNBC cells migration and invasion in vivo and in vitro. LDL induces higher mitochondrial mass and network spread in migrating cells, in an actin remodeling-dependent manner, and transcriptomic and energetic analyses revealed that LDL renders TNBC cells dependent on fatty acids (FA) usage for mitochondrial respiration. Indeed, engagement on FA transport into the mitochondria is required for LDL-induced migration and mitochondrial remodeling. Mechanistically, LDL treatment leads to mitochondrial long-chain fatty acid accumulation and increased reactive oxygen species (ROS) production. Importantly, CD36 or ROS blockade abolished LDL-induced cell migration and mitochondria metabolic adaptations. Our data suggest that LDL induces TNBC cells migration by reprogramming mitochondrial metabolism, revealing a new vulnerability in metastatic breast cancer.Significance: LDL induces breast cancer cell migration that relies on CD36 for mitochondrial metabolism and network remodeling, providing an antimetastatic metabolic strategy.
  6. Front Cell Dev Biol. 2023 ;11 1082213
      Introduction: The mitochondrial uniporter (MCU) Ca2+ ion channel represents the primary means for Ca2+ uptake by mitochondria. Mitochondrial matrix Ca2+ plays critical roles in mitochondrial bioenergetics by impinging upon respiration, energy production and flux of biochemical intermediates through the TCA cycle. Inhibition of MCU in oncogenic cell lines results in an energetic crisis and reduced cell proliferation unless media is supplemented with nucleosides, pyruvate or α-KG. Nevertheless, the roles of MCU-mediated Ca2+ influx in cancer cells remain unclear, in part because of a lack of genetic models. Methods: MCU was genetically deleted in transformed murine fibroblasts for study in vitro and in vivo. Tumor formation and growth were studied in murine xenograft models. Proliferation, cell invasion, spheroid formation and cell cycle progression were measured in vitro. The effects of MCU deletion on survival and cell-death were determined by probing for live/death markers. Mitochondrial bioenergetics were studied by measuring mitochondrial matrix Ca2+ concentration, membrane potential, global dehydrogenase activity, respiration, ROS production and inactivating-phosphorylation of pyruvate dehydrogenase. The effects of MCU rescue on metabolism were examined by tracing of glucose and glutamine utilization for fueling of mitochondrial respiration. Results: Transformation of primary fibroblasts in vitro was associated with increased MCU expression, enhanced MCU-mediated Ca2+ uptake, altered mitochondrial matrix Ca2+ concentration responses to agonist stimulation, suppression of inactivating-phosphorylation of pyruvate dehydrogenase and a modest increase of mitochondrial respiration. Genetic MCU deletion inhibited growth of HEK293T cells and transformed fibroblasts in mouse xenograft models, associated with reduced proliferation and delayed cell-cycle progression. MCU deletion inhibited cancer stem cell-like spheroid formation and cell invasion in vitro, both predictors of metastatic potential. Surprisingly, mitochondrial matrix [Ca2+], membrane potential, global dehydrogenase activity, respiration and ROS production were unaffected. In contrast, MCU deletion elevated glycolysis and glutaminolysis, strongly sensitized cell proliferation to glucose and glutamine limitation, and altered agonist-induced cytoplasmic Ca2+ signals. Conclusion: Our results reveal a dependence of tumorigenesis on MCU, mediated by a reliance on MCU for cell metabolism and Ca2+ dynamics necessary for cell-cycle progression and cell proliferation.
    Keywords:  calcium; cancer; metabolism; mitochondria; stable isotope tracing; uniporter
  7. Oncogene. 2023 Jun 24.
      Therapy resistance to second-generation androgen receptor (AR) antagonists, such as enzalutamide, is common in patients with advanced prostate cancer (PCa). To understand the metabolic alterations involved in enzalutamide resistance, we performed metabolomic, transcriptomic, and cistromic analyses of enzalutamide-sensitive and -resistant PCa cells, xenografts, patient-derived organoids, patient-derived explants, and tumors. We noted dramatically higher basal and inducible levels of reactive oxygen species (ROS) in enzalutamide-resistant PCa and castration-resistant PCa (CRPC), in comparison to enzalutamide-sensitive PCa cells or primary therapy-naive tumors respectively. Unbiased metabolomic evaluation identified that glutamine metabolism was consistently upregulated in enzalutamide-resistant PCa cells and CRPC tumors. Stable isotope tracing studies suggest that this enhanced glutamine metabolism drives an antioxidant program that allows these cells to tolerate higher basal levels of ROS. Inhibition of glutamine metabolism with either a small-molecule glutaminase inhibitor or genetic knockout of glutaminase enhanced ROS levels, and blocked the growth of enzalutamide-resistant PCa. The critical role of compensatory antioxidant pathways in maintaining enzalutamide-resistant PCa cells was validated by targeting another antioxidant program driver, ferredoxin 1. Taken together, our data identify a metabolic need to maintain antioxidant programs and a potentially targetable metabolic vulnerability in enzalutamide-resistant PCa.
  8. Nat Cell Biol. 2023 Jun 29.
      Fasting triggers diverse physiological adaptations including increases in circulating fatty acids and mitochondrial respiration to facilitate organismal survival. The mechanisms driving mitochondrial adaptations and respiratory sufficiency during fasting remain incompletely understood. Here we show that fasting or lipid availability stimulates mTORC2 activity. Activation of mTORC2 and phosphorylation of its downstream target NDRG1 at serine 336 sustains mitochondrial fission and respiratory sufficiency. Time-lapse imaging shows that NDRG1, but not the phosphorylation-deficient NDRG1Ser336Ala mutant, engages with mitochondria to facilitate fission in control cells, as well as in those lacking DRP1. Using proteomics, a small interfering RNA screen, and epistasis experiments, we show that mTORC2-phosphorylated NDRG1 cooperates with small GTPase CDC42 and effectors and regulators of CDC42 to orchestrate fission. Accordingly, RictorKO, NDRG1Ser336Ala mutants and Cdc42-deficient cells each display mitochondrial phenotypes reminiscent of fission failure. During nutrient surplus, mTOR complexes perform anabolic functions; however, paradoxical reactivation of mTORC2 during fasting unexpectedly drives mitochondrial fission and respiration.
  9. Nat Genet. 2023 Jun 29.
      Pathogenic mutations in mitochondrial DNA (mtDNA) compromise cellular metabolism, contributing to cellular heterogeneity and disease. Diverse mutations are associated with diverse clinical phenotypes, suggesting distinct organ- and cell-type-specific metabolic vulnerabilities. Here we establish a multi-omics approach to quantify deletions in mtDNA alongside cell state features in single cells derived from six patients across the phenotypic spectrum of single large-scale mtDNA deletions (SLSMDs). By profiling 206,663 cells, we reveal the dynamics of pathogenic mtDNA deletion heteroplasmy consistent with purifying selection and distinct metabolic vulnerabilities across T-cell states in vivo and validate these observations in vitro. By extending analyses to hematopoietic and erythroid progenitors, we reveal mtDNA dynamics and cell-type-specific gene regulatory adaptations, demonstrating the context-dependence of perturbing mitochondrial genomic integrity. Collectively, we report pathogenic mtDNA heteroplasmy dynamics of individual blood and immune cells across lineages, demonstrating the power of single-cell multi-omics for revealing fundamental properties of mitochondrial genetics.
  10. Cancer Res Commun. 2023 Jun;3(6): 1041-1056
      Glioblastomas (GBM) are heterogeneous tumors with high metabolic plasticity. Their poor prognosis is linked to the presence of glioblastoma stem cells (GSC), which support resistance to therapy, notably to temozolomide (TMZ). Mesenchymal stem cells (MSC) recruitment to GBM contributes to GSC chemoresistance, by mechanisms still poorly understood. Here, we provide evidence that MSCs transfer mitochondria to GSCs through tunneling nanotubes, which enhances GSCs resistance to TMZ. More precisely, our metabolomics analyses reveal that MSC mitochondria induce GSCs metabolic reprograming, with a nutrient shift from glucose to glutamine, a rewiring of the tricarboxylic acid cycle from glutaminolysis to reductive carboxylation and increase in orotate turnover as well as in pyrimidine and purine synthesis. Metabolomics analysis of GBM patient tissues at relapse after TMZ treatment documents increased concentrations of AMP, CMP, GMP, and UMP nucleotides and thus corroborate our in vitro analyses. Finally, we provide a mechanism whereby mitochondrial transfer from MSCs to GSCs contributes to GBM resistance to TMZ therapy, by demonstrating that inhibition of orotate production by Brequinar (BRQ) restores TMZ sensitivity in GSCs with acquired mitochondria. Altogether, these results identify a mechanism for GBM resistance to TMZ and reveal a metabolic dependency of chemoresistant GBM following the acquisition of exogenous mitochondria, which opens therapeutic perspectives based on synthetic lethality between TMZ and BRQ.Significance: Mitochondria acquired from MSCs enhance the chemoresistance of GBMs. The discovery that they also generate metabolic vulnerability in GSCs paves the way for novel therapeutic approaches.
  11. Cancer Res Commun. 2023 Jun;3(6): 1067-1077
      The arginine methyltransferase CARM1 exhibits high expression levels in several human cancers, with the trend also observed in ovarian cancer. However, therapeutic approaches targeting tumors that overexpress CARM1 have not been explored. Cancer cells exploit metabolic reprogramming such as fatty acids for their survival. Here we report that CARM1 promotes monounsaturated fatty acid synthesis and fatty acid reprogramming represents a metabolic vulnerability for CARM1-expressing ovarian cancer. CARM1 promotes the expression of genes encoding rate-limiting enzymes of de novo fatty acid metabolism such as acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FASN). In addition, CARM1 upregulates stearoyl-CoA desaturase 1 (SCD1) that produces monounsaturated fatty acid by desaturation. Thus, CARM1 enhances de novo fatty acids synthesis which was subsequently utilized for synthesis of monounsaturated fatty acids. Consequently, inhibition of SCD1 suppresses the growth of ovarian cancer cells in a CARM1 status-dependent manner, which was rescued by the addition of monounsaturated fatty acids. Consistently, CARM1-expressing cells were more tolerant to the addition of saturated fatty acids. Indeed, SCD1 inhibition demonstrated efficacy against ovarian cancer in both orthotopic xenograft and syngeneic mouse models in a CARM1-dependent manner. In summary, our data show that CARM1 reprograms fatty acid metabolism and targeting SCD1 through pharmacological inhibition can serve as a potent therapeutic approach for CARM1-expressing ovarian cancers.Significance: CARM1 reprograms fatty acid metabolism transcriptionally to support ovarian cancer growth by producing monounsaturated fatty acids, supporting SCD1 inhibition as a rational strategy for treating CARM1-expressing ovarian cancer.
  12. Acta Physiol (Oxf). 2023 Jun 27. e14016
      The mitochondrial pyruvate carrier (MPC) resides in the mitochondrial inner membrane, where it links cytosolic and mitochondrial metabolism by transporting pyruvate produced in glycolysis into the mitochondrial matrix. Due to its central metabolic role, it has been proposed as a potential drug target for diabetes, non-alcoholic fatty liver disease, neurodegeneration, and cancers relying on mitochondrial metabolism. Little is known about the structure and mechanism of MPC, as the proteins involved were only identified a decade ago and technical difficulties concerning their purification and stability have hindered progress in functional and structural analyses. The functional unit of MPC is a hetero-dimer comprising two small homologous membrane proteins, MPC1/MPC2 in humans, with the alternative complex MPC1L/MPC2 forming in the testis, but MPC proteins are found throughout the tree of life. The predicted topology of each protomer consists of an amphipathic helix followed by three transmembrane helices. An increasing number of inhibitors are being identified, expanding MPC pharmacology and providing insights into the inhibitory mechanism. Here, we provide critical insights on the composition, structure, and function of the complex and we summarize the different classes of small molecule inhibitors and their potential in therapeutics.
    Keywords:  metabolism; mitochondria; pyruvate transport; small molecule inhibitors; transport mechanism
  13. Nat Commun. 2023 Jun 28. 14(1): 3823
      Pancreatic Ductal Adenocarcinoma (PDAC) is highly resistant to chemotherapy. Effective alternative therapies have yet to emerge, as chemotherapy remains the best available systemic treatment. However, the discovery of safe and available adjuncts to enhance chemotherapeutic efficacy can still improve survival outcomes. We show that a hyperglycemic state substantially enhances the efficacy of conventional single- and multi-agent chemotherapy regimens against PDAC. Molecular analyses of tumors exposed to high glucose levels reveal that the expression of GCLC (glutamate-cysteine ligase catalytic subunit), a key component of glutathione biosynthesis, is diminished, which in turn augments oxidative anti-tumor damage by chemotherapy. Inhibition of GCLC phenocopies the suppressive effect of forced hyperglycemia in mouse models of PDAC, while rescuing this pathway mitigates anti-tumor effects observed with chemotherapy and high glucose.
  14. J Clin Invest. 2023 Jun 29. pii: e167651. [Epub ahead of print]
      Despite the success of KRAS G12C inhibitors in non-small cell lung cancer (NSCLC), more effective treatments are needed. One preclinical strategy has been to co-target RAS and mTOR pathways, however toxicity due to broad mTOR inhibition has limited its utility. Therefore, we sought to develop a more refined means of targeting cap-dependent translation and identify the most therapeutically important eIF4F-translated targets. Here we show that an eIF4A inhibitor, which targets a component of eIF4F, dramatically enhances the effects of KRAS G12C inhibitors in NSCLCs and together these agents induce potent tumor regression in vivo. By screening a broad panel of eIF4F targets, we show that this cooperativity is driven by effects on BCL-2 family proteins. Moreover, because multiple BCL-2 family members are concomitantly suppressed, these agents are broadly efficacious in NSCLCs, irrespective of their dependency on MCL1, BCL-xL, or BCL-2, which is known to be heterogeneous. Finally, we show that MYC overexpression confers sensitivity to this combination because it creates a dependency on eIF4A for BCL-2 family protein expression. Together, these studies identify a promising therapeutic strategy for KRAS-mutant NSCLCs, demonstrate that BCL-2 proteins are the key mediators of the therapeutic response in this tumor type, and uncover a predictive biomarker of sensitivity.
    Keywords:  Drug therapy; Lung cancer; Oncology; Signal transduction
  15. Mol Biol Cell. 2023 Jun 28. mbcE23050205
      Almost all mitochondrial proteins are synthesized in the cytosol and subsequently targeted to mitochondria. The accumulation of non-imported precursor proteins occurring upon mitochondrial dysfunction can challenge cellular protein homeostasis. Here we show that blocking protein translocation into mitochondria results in the accumulation of mitochondrial membrane proteins at the endoplasmic reticulum, thereby triggering the unfolded protein response (UPRER). Moreover, we find that mitochondrial membrane proteins are also routed to the ER under physiological conditions. The level of ER-resident mitochondrial precursors is enhanced by import defects as well as metabolic stimuli that increase the expression of mitochondrial proteins. Under such conditions, the UPRER is crucial to maintain protein homeostasis and cellular fitness. We propose the ER serves as a physiological buffer zone for those mitochondrial precursors that cannot be immediately imported into mitochondria while engaging the UPRER to adjust the ER proteostasis capacity to the extent of precursor accumulation.
  16. J Med Chem. 2023 Jun 26.
      Discovery of small molecule inhibitors targeting Mcl-1 (Myeloid cell leukemia 1) confronts many challenges. Based on the fact that Mcl-1 is mainly localized in mitochondria, we propose a new strategy of targeting mitochondria to improve the binding efficiency of Mcl-1 inhibitors. We report the discovery of complex 9, the first mitochondrial targeting platinum-based inhibitor of Mcl-1, which selectively binds to Mcl-1 with high binding affinity. Complex 9 was mainly concentrated in the mitochondria of tumor cells which led to an enhanced antitumor efficacy. Complex 9 induced Bax/Bak-dependent apoptosis in LP-1 cells and synergized with ABT-199 to kill ABT-199 resistant cells in multiple cancer models. Complex 9 was effective and tolerable as a single agent or in combination with ABT-199 in mouse models. This research work demonstrated that developing mitochondria-targeting Mcl-1 inhibitors is a new potentially efficient strategy for tumor therapy.
  17. Biosens Bioelectron. 2023 May 11. pii: S0956-5663(23)00310-X. [Epub ahead of print]237 115368
      The detection and comparison of the amount of superoxide anion (O2.-) released by different complexes in mitochondrial electron transport chain (ETC) can locate the main electron leakage sites in mitochondria. In order to realize this, we designed an ultrasensitive photoelectrochemical (PEC) sensor by in situ hydrothermal growth of MnO2 nanosheets on Co3O4 nanowires array modified Ti substrate (NWA|Ti). Due to the formation of a core-shell p-n heterojunction with high specific surface area, tight surface contact and plentiful oxygen vacancies (OVs), MnO2@Co3O4 NWA|Ti possesses a strong visible light absorption, high charges transfer and separation ability. The proposed PEC sensor exhibited a wide linear range of 0.1-50000 nM and a low detection limit of 0.025 nM towards H2O2. Due to the rapid conversion of O2.- to H2O2 inside mitochondria, the PEC sensor can indirectly monitor the electron leakage in the ETC. Specifically, four selected mitochondrial inhibitors specifically inhibited the corresponding complex in mitochondria extracted from living HepG2 cells (hepatocellular carcinoma cells), and the H2O2 levels converted from O2.- was measured by the PEC sensor. It is evident that IQ (ubiquinone binding) site of complex I and Qo (ubiquinol oxidation) site of complex III are the key sites at which electron leakage occurred. This study could provide meaningful information for the diagnosis and treatment of certain disease caused by oxidative stress due to the electron leakage.
    Keywords:  Core-shell p-n heterojunction; HepG2 cells; Mitochondrial inhibitors; Photoelectrochemical sensor; Superoxide anion
  18. Sci Rep. 2023 06 28. 13(1): 10497
      Glioblastoma, a malignant tumor, has no curative treatment. Recently, mitochondria have been considered a potential target for treating glioblastoma. Previously, we reported that agents initiating mitochondrial dysfunction were effective under glucose-starved conditions. Therefore, this study aimed to develop a mitochondria-targeted treatment to achieve normal glucose conditions. This study used U87MG (U87), U373, and patient-derived stem-like cells as well as chloramphenicol (CAP) and 2-deoxy-D-glucose (2-DG). We investigated whether CAP and 2-DG inhibited the growth of cells under normal and high glucose concentrations. In U87 cells, 2-DG and long-term CAP administration were more effective under normal glucose than high-glucose conditions. In addition, combined CAP and 2-DG treatment was significantly effective under normal glucose concentration in both normal oxygen and hypoxic conditions; this was validated in U373 and patient-derived stem-like cells. 2-DG and CAP acted by influencing iron dynamics; however, deferoxamine inhibited the efficacy of these agents. Thus, ferroptosis could be the underlying mechanism through which 2-DG and CAP act. In conclusion, combined treatment of CAP and 2-DG drastically inhibits cell growth of glioblastoma cell lines even under normal glucose conditions; therefore, this treatment could be effective for glioblastoma patients.
  19. Immunopharmacol Immunotoxicol. 2023 Jun 29. 1-12
      BACKGROUND: Multiple targets of chimeric antigen receptor T cells (CAR-T cells) are shared expressed by tumor cells and T cells, these self antigens may stimulate CAR-T cells continuously during the expansion. Persistent exposure to antigens is considered to cause metabolic reprogramming of T cells and the metabolic profiling is critical in determining the cell fate and effector function of CAR-T cells. However, whether self-antigen stimulation during CAR-T cell generation could remodel the metabolic profiling is unclear. In this study, we aim to investigate the metabolic characteristics of CD26 CAR-T cells, which expressed CD26 antigens themselves.METHODS: The mitochondrial biogenesis of CD26 and CD19 CAR-T cells during expansion was evaluated by the mitochondrial content, mitochondrial DNA copy numbers and genes involved in mitochondrial regulation. The metabolic profiling was investigated by the ATP production, mitochondrial quality and the expression of metabolism-related genes. Furthermore, we assessed the phenotypes of CAR-T cells through memory-related markers.
    RESULTS: We reported that CD26 CAR-T cells had elevated mitochondrial biogenesis, ATP production and oxidative phosphorylation at early expansion stage. However, the mitochondrial biogenesis, mitochondrial quality, oxidative phosphorylation and glycolytic activity were all weakened at later expansion stage. On the contrary, CD19 CAR-T cells did not exhibit such characteristics.
    CONCLUSION: CD26 CAR-T cells showed distinctive metabolic profiling during expansion that was extremely unfavorable to cell persistence and function. These findings may provide new insights for the optimization of CD26 CAR-T cells in terms of metabolism.
    Keywords:  CD19 CAR-T cells; CD26 CAR-T cells; glycolysis; metabolism; mitochondrial biogenesis
  20. Cancer Discov. 2023 Jun 26. pii: CD-22-1297. [Epub ahead of print]
      The BCL-2 inhibitor venetoclax has recently emerged as an important component of acute myeloid leukemia (AML) therapy. Notably, use of this agent has revealed a previously unrecognized form of pathogenesis characterized by monocytic disease progression. We demonstrate that this form of disease arises from a fundamentally different type of leukemia stem cell (LSC), which we designate as monocytic LSC (m-LSC), that is developmentally and clinically distinct from the more well-described primitive LSC (p-LSC). The m-LSC is distinguished by a unique immunophenotype (CD34-, CD4+, CD11b-, CD14-, CD36-), unique transcriptional state, reliance on purine metabolism, and selective sensitivity to cladribine. Critically, in some instances m-LSC and p-LSC subtypes can co-reside in the same AML patient and simultaneously contribute to overall tumor biology. Thus, our findings demonstrate that LSC heterogeneity has direct clinical significance and highlights the need to distinguish and target m-LSCs as a means to improve clinical outcomes with venetoclax-based regimens.
  21. J Cell Mol Med. 2023 Jun 29.
      Few approaches have been conducted in the treatment of renal cell carcinoma (RCC) after nephrectomy, resulting in a high mortality rate in urological tumours. Mitophagy is a mechanism of mitochondrial quality control that enables selective degradation of damaged and unnecessary mitochondria. Previous studies have found that glycerol-3-phosphate dehydrogenase 1-like (GPD1L) is associated with the progression of tumours such as lung cancer, colorectal cancer and oropharyngeal cancer, but the potential mechanism in RCC is still unclear. In this study, microarrays from tumour databases were analysed. The expression of GPD1L was confirmed by RT-qPCR and western blotting. The effect and mechanism of GPD1L were explored using cell counting kit 8, wound healing, invasion, flow cytometry and mitophagy-related experiments. The role of GPD1L was further confirmed in vivo. The results showed that GPD1L expression was downregulated and positively correlated with prognosis in RCC. Functional experiments revealed that GPD1L prevented proliferation, migration and invasion while promoting apoptosis and mitochondrial injury in vitro. The mechanistic results indicated that GPD1L interacted with PINK1, promoting PINK1/Parkin-mediated mitophagy. However, inhibition of PINK1 reversed GPD1L-mediated mitochondrial injury and mitophagy. Moreover, GPD1L prevented tumour growth and promoted mitophagy by activating the PINK1/Parkin pathway in vivo. Our study shows that GPD1L has a positive correlation with the prognosis of RCC. The potential mechanism involves interacting with PINK1 and regulating the PINK1/Parkin pathway. In conclusion, these results reveal that GPD1L can act as a biomarker and target for RCC diagnosis and therapy.
    Keywords:  PINK1/Parkin pathway; mitophagy; phosphate dehydrogenase 1-like; renal cell carcinoma
  22. Cancers (Basel). 2023 Jun 17. pii: 3225. [Epub ahead of print]15(12):
      An in vitro culture period of at least 2 weeks is required to produce sufficient natural killer (NK) cells for immunotherapy, which are the key effectors in hematological malignancy treatment. Mitochondrial damage and fragmentation reduce the NK cell immune surveillance capacity. Thus, we hypothesized that the transfer of healthy mitochondria to NK cells could enhance their anticancer effects. Allogeneic healthy mitochondria isolated from WRL-68 cells were transferred to NK cells. We evaluated NK cells' proliferative capacity, cell cycle, and cytotoxic capacity against various cancer cell types by analyzing specific lysis and the cytotoxic granules released. The relationship between the transferred allogenic mitochondrial residues and NK cell function was determined. After mitochondrial transfer, the NK cell proliferation rate was 1.2-fold higher than that of control cells. The mitochondria-treated NK cells secreted a 2.7-, 4.1-, and 5-fold higher amount of granzyme B, perforin, and IFN-γ, respectively, when co-cultured with K562 cells. The specific lysis of various solid cancer cells increased 1.3-1.6-fold. However, once allogeneic mitochondria were eliminated, the NK cell activity returned to the pre-mitochondrial transfer level. Mitochondria-enriched NK cells have the potential to be used as a novel solid cancer treatment agent, without the need for in vitro cytokine-induced culture.
    Keywords:  anticancer immunotherapy; immune cell therapy; mitochondria; mitochondrial transfer; natural killer cells
  23. Free Radic Res. 2023 Jun 26. 1-31
      Cancer cells exhibit an altered redox balance and aberrant redox signaling due to genetic, metabolic, and microenvironment-associated reprogramming. Persistently elevated levels of reactive oxygen species (ROS) contribute to many aspects of tumor development and progression. Emerging studies demonstrated the vital role of apurinic/apyrimidinic endonuclease 1 or reduction/oxidation (redox) factor 1(APE1/Ref-1) in the oxidative stress response and survival of cancer cells. APE1/Ref-1 is a multifunctional enzyme involved in the DNA damage response and functions as a redox regulator of transcription factors. We herein demonstrated that basal hydrogen peroxide (H2O2) and APE1/Ref-1 expression levels were markedly higher in cancer cell lines than in non-cancerous cells. Elevated APE1/Ref-1 levels were associated with shorter survival in liver cancer patients. Mechanistically, we showed that H2O2 activated nuclear factor-κB (NF-κB). RelA/p65 inhibited the expression of the E3 ubiquitin ligase Parkin, possibly by interfering with ATF4 activity. Parkin was responsible for the ubiquitination and proteasomal degradation of APE1/Ref-1; therefore, the H2O2-induced suppression of Parkin expression increased APE1/Ref-1 levels. The probability of survival was lower in liver cancer patients with low Parkin and high RelA expression levels. Additionally, Parkin and RelA expression levels negatively and positively correlated with APE1/Ref-1 levels, respectively, in the TCGA liver cancer cohort. We concluded that increases in APE1/Ref-1 via the NF-κB and Parkin pathways are critical for cancer cell survival under oxidative stress. The present results show the potential of the NF-κB-Parkin-APE1/Ref-1 axis as a prognostic factor and therapeutic strategy to eradicate liver cancer.
    Keywords:  APE1/Ref-1; NF-κB; Parkin; hydrogen peroxide; liver cancer
  24. Nat Commun. 2023 Jun 30. 14(1): 3877
      DNA derived from chemotherapeutics-killed tumor cells is one of the most important damage-associated molecular patterns that can activate the cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) pathway in antigen-presenting cells (APCs) and promote antitumor immunity. However, conventional chemotherapy displays limited tumor cell killing and ineffective transfer of stable tumor DNA to APCs. Here we show that liposomes loaded with an optimized ratio of indocyanine green and doxorubicin, denoted as LID, efficiently generate reactive oxygen species upon exposure to ultrasound. LID plus ultrasound enhance the nuclear delivery of doxorubicin, induce tumor mitochondrial DNA oxidation, and promote oxidized tumor mitochondrial DNA transfer to APCs for effective activation of cGAS-STING signaling. Depleting tumor mitochondrial DNA or knocking out STING in APCs compromises the activation of APCs. Furthermore, systemic injection of LID plus ultrasound over the tumor lead to targeted cytotoxicity and STING activation, eliciting potent antitumor T cell immunity, which upon the combination with immune checkpoint blockade leads to regression of bilateral MC38, CT26, and orthotopic 4T1 tumors in female mice. Our study sheds light on the importance of oxidized tumor mitochondrial DNA in STING-mediated antitumor immunity and may inspire the development of more effective strategies for cancer immunotherapy.
  25. Exp Dermatol. 2023 Jun 28.
      The dysregulation of branched-chain amino acid (BCAA) metabolism and related enzymes has been greatly implicated in the progression of multiple types of cancer, whereas remains far from understood in melanoma. Here, we explored the role of the BCAA metabolism enzyme BCKDHA in melanoma pathogenesis and elucidated the underlying mechanisms. In vitro cell biology experiments and in vivo pre-clinical mice model experiments were performed to investigate the role of BCKDHA in melanoma progression. RNA sequencing, immunohistochemical/immunofluorescence staining and bioinformatics analysis were used to examine the underlying mechanism. BCKDHA expression was prominently increased in both melanoma tissues and cell lines. The up-regulation of BCKDHA promoted long-term tumour cell proliferation, invasion and migration in vitro and tumour growth in vivo. Through RNA-sequencing technology, it was found that BCKDHA regulated the expressions of lipogenic fatty acid synthase (FASN) and ATP-citrate lyase (ACLY), which was thereafter proved to mediate the oncogenic role of BCKDHA in melanoma. Our results demonstrate that BCKDHA promotes melanoma progression by regulating FASN and ACLY expressions. Targeting BCKDHA could be exploited as a promising strategy to restrain tumour progression in melanoma.
    Keywords:  ACLY; BCKDHA; FASN; lipogenesis; melanoma
  26. Cancer Res Commun. 2023 Jun;3(6): 969-979
      Ribosomes in cancer cells accumulate numerous patient-specific structural and functional modifications that facilitate tumor progression by modifying protein translation. We have taken a unique synthetic chemistry approach to generate novel macrolides, Ribosome modulating agents (RMA), that are proposed to act distal to catalytic sites and exploit cancer ribosome heterogeneity. The RMA ZKN-157 shows two levels of selectivity: (i) selective translation inhibition of a subset of proteins enriched for components of the ribosome and protein translation machinery that are upregulated by MYC; and (ii) selective inhibition of proliferation of a subset of colorectal cancer cell lines. Mechanistically, the selective ribosome targeting in sensitive cells triggered cell-cycle arrest and apoptosis. Consequently, in colorectal cancer, sensitivity to ZKN-157 in cell lines and patient-derived organoids was restricted to the consensus molecular subtype 2 (CMS2) subtype that is distinguished by high MYC and WNT pathway activity. ZKN-157 showed efficacy as single agent and, the potency and efficacy of ZKN-157 synergized with clinically approved DNA-intercalating agents which have previously been shown to inhibit ribogenesis as well. ZKN-157 thus represents a new class of ribosome modulators that display cancer selectivity through specific ribosome inhibition in the CMS2 subtype of colorectal cancer potentially targeting MYC-driven addiction to high protein translation.Significance: This study demonstrates that ribosome heterogeneity in cancer can be exploited to develop selective ribogenesis inhibitors. The colorectal cancer CMS2 subtype, with a high unmet need for therapeutics, shows vulnerability to our novel selective ribosome modulator. The mechanism suggests that other cancer subtypes with high MYC activation could also be targeted.
  27. Biomolecules. 2023 05 23. pii: 876. [Epub ahead of print]13(6):
      The antiapoptotic protein Bcl-xL is a major regulator of cell death and survival, but many aspects of its functions remain elusive. It is mostly localized in the mitochondrial outer membrane (MOM) owing to its C-terminal hydrophobic α-helix. In order to gain further information about its membrane organization, we set up a model system combining cell-free protein synthesis and nanodisc insertion. We found that, contrary to its proapoptotic partner Bax, neosynthesized Bcl-xL was spontaneously inserted into nanodiscs. The deletion of the C-terminal α-helix of Bcl-xL prevented nanodisc insertion. We also found that nanodisc insertion protected Bcl-xL against the proteolysis of the 13 C-terminal residues that occurs during expression of Bcl-xL as a soluble protein in E. coli. Interestingly, we observed that Bcl-xL increased the insertion of Bax into nanodiscs, in a similar way to that which occurs in mitochondria. Cell-free synthesis in the presence of nanodiscs is, thus, a suitable model system to study the molecular aspects of the interaction between Bcl-xL and Bax during their membrane insertion.
    Keywords:  Bax; Bcl-xL; MALDI; apoptosis; cell-free synthesis; membrane insertion; nanodiscs
  28. Geroscience. 2023 Jun 27.
      Mitochondrial improvements resulting from behavioral interventions, such as diet and exercise, are systemic and apparent across multiple tissues. Here, we test the hypothesis that factors present in serum, and therefore circulating throughout the body, can mediate changes in mitochondrial function in response to intervention. To investigate this, we used stored serum from a clinical trial comparing resistance training (RT) and RT plus caloric restriction (RT + CR) to examine effects of blood borne circulating factors on myoblasts in vitro. We report that exposure to dilute serum is sufficient to mediate bioenergetic benefits of these interventions. Additionally, serum-mediated bioenergetic changes can differentiate between interventions, recapitulate sex differences in bioenergetic responses, and is linked to improvements in physical function and inflammation. Using metabolomics, we identified circulating factors associated with changes in mitochondrial bioenergetics and the effects of interventions. This study provides new evidence that circulating factors play a role in the beneficial effects of interventions that improve healthspan among older adults. Understanding the factors that drive improvements in mitochondrial function is a key step towards predicting intervention outcomes and developing strategies to countermand systemic age-related bioenergetic decline.
    Keywords:  Bioenergetics; Caloric restriction; Metabolomics; Mitochondria; Older adults; Resistance training
  29. Front Immunol. 2023 ;14 1189953
      Immune therapies targeting the PD-1/PD-L1 pathway have been employed in the treatment of breast cancer, which requires aerobic glycolysis to sustain breast cancer cells growth. However, whether PD-L1 expression is regulated by glycolysis in breast cancer cells remains to be further elucidated. Here, we demonstrate that glycolytic enzyme hexokinase 2 (HK2) plays a crucial role in upregulating PD-L1 expression. Under high glucose conditions, HK2 acts as a protein kinase and phosphorylates IκBα at T291 in breast cancer cells, leading to the rapid degradation of IκBα and activation of NF-κB, which enters the nucleus and promotes PD-L1 expression. Immunohistochemistry staining of human breast cancer specimens and bioinformatics analyses reveals a positive correlation between HK2 and PD-L1 expression levels, which are inversely correlated with immune cell infiltration and survival time of breast cancer patients. These findings uncover the intrinsic and instrumental connection between aerobic glycolysis and PD-L1 expression-mediated tumor cell immune evasion and underscore the potential to target the protein kinase activity of HK2 for breast cancer treatment.
    Keywords:  HK2; IκBα; NF-κB; PD-L1; breast cancer; immunotherapy; metabolism
  30. Nutrients. 2023 Jun 13. pii: 2730. [Epub ahead of print]15(12):
      The ketogenic diet (KD) is hypothesized to impact tumor progression by altering tumor metabolism. In this study, we assessed the impact of an unrestricted KD on epithelial ovarian cancer (EOC) tumor growth, gene expression, and metabolite concentration in a mouse model. ID8 EOC cells, which were syngeneic with C57Bl/6J mouse strain and transfected with luciferase (ID8-luc), were injectedand monitored for tumor development. Female mice were fed either a strict KD, a high fat/low carbohydrate (HF/LC) diet, or a low fat/high carbohydrate (LF/HC) diet (n = 10 mice per group) ad libitum. EOC tumor growth was monitored weekly, and tumor burden was determined based on luciferase fluorescence (photons/second). At the endpoint (42 days), tumors were collected and processed for RNA sequencing. Plasma and tumor metabolites were evaluated using LC-MS. The KD-fed mice exhibited a statistically significant increase in tumor progression in comparison to the HF/LC- and LF/HC-fed groups (9.1 vs. 2.0 vs. 3.1-fold, respectively, p < 0.001). The EOC tumors of the KD-fed mice exhibited significant enrichment of the peroxisome proliferator-activated receptor (PPAR) signaling and fatty acid metabolism pathways based on the RNA sequencing analysis when compared to the LF/HC- and HF/LC-fed mice. Thus, unrestricted KD diet enhanced tumor progression in our mouse EOC model. KD was associated with the upregulation of fatty acid metabolism and regulation pathways, as well as enrichment of fatty acid and glutamine metabolites.
    Keywords:  RNA sequencing; dietary intervention; high fat diet; ketone bodies; mice; ovarian cancer; tumor metabolism; tumor progression
  31. Mol Cancer Res. 2023 Jun 26. pii: MCR-22-0935. [Epub ahead of print]
      Correlations between the oxidative stress response and metabolic reprogramming have been observed during malignant tumor formation; however, the detailed mechanism remains elusive. The transcription factor Nrf2, a master regulator of the oxidative stress response, mediates metabolic reprogramming in multiple cancers. In a mouse model of hepatocellular carcinoma (HCC), through metabolic profiling, genome-wide gene expression, and chromatin structure analyses, we present new evidence showing that in addition to altering antioxidative stress response signaling, Nrf2 ablation impairs multiple metabolic pathways to reduce the generation of acetyl-CoA and suppress histone acetylation in tumors, but not in tumor-adjacent normal tissue. Nrf2 ablation and dysregulated histone acetylation impair transcription complex assembly on downstream target antioxidant and metabolic regulatory genes for expression regulation. Mechanistic studies indicate that the regulatory function of Nrf2 is low glucose dependent, the effect of which is demolished under energy refeeding. Together, our results implicate an unexpected effect of Nrf2 on acetyl-CoA generation, in addition to its classic antioxidative stress response regulatory activity, integrates metabolic and epigenetic programs to drive HCC progression. Implications: This study highlights that Nrf2 integrates metabolic and epigenetic regulatory networks to dictate tumor progression and that Nrf2 targeting is therapeutically exploitable in HCC treatment.
  32. Nature. 2023 Jun 28.
      In metazoan organisms, cell competition acts as a quality control mechanism to eliminate unfit cells in favour of their more robust neighbours1,2. This mechanism has the potential to be maladapted, promoting the selection of aggressive cancer cells3-6. Tumours are metabolically active and are populated by stroma cells7,8, but how environmental factors affect cancer cell competition remains largely unknown. Here we show that tumour-associated macrophages (TAMs) can be dietarily or genetically reprogrammed to outcompete MYC-overexpressing cancer cells. In a mouse model of breast cancer, MYC overexpression resulted in an mTORC1-dependent 'winner' cancer cell state. A low-protein diet inhibited mTORC1 signalling in cancer cells and reduced tumour growth, owing unexpectedly to activation of the transcription factors TFEB and TFE3 and mTORC1 in TAMs. Diet-derived cytosolic amino acids are sensed by Rag GTPases through the GTPase-activating proteins GATOR1 and FLCN to control Rag GTPase effectors including TFEB and TFE39-14. Depletion of GATOR1 in TAMs suppressed the activation of TFEB, TFE3 and mTORC1 under the low-protein diet condition, causing accelerated tumour growth; conversely, depletion of FLCN or Rag GTPases in TAMs activated TFEB, TFE3 and mTORC1 under the normal protein diet condition, causing decelerated tumour growth. Furthermore, mTORC1 hyperactivation in TAMs and cancer cells and their competitive fitness were dependent on the endolysosomal engulfment regulator PIKfyve. Thus, noncanonical engulfment-mediated Rag GTPase-independent mTORC1 signalling in TAMs controls competition between TAMs and cancer cells, which defines a novel innate immune tumour suppression pathway that could be targeted for cancer therapy.
  33. ACS Synth Biol. 2023 Jun 27.
      ATP is the energy currency of the cell and new methods for ATP regeneration will benefit a range of emerging biotechnology applications including synthetic cells. We designed and assembled a membraneless ATP-regenerating enzymatic cascade by exploiting the substrate specificities of selected NAD(P)(H)-dependent oxidoreductases combined with substrate-specific kinases. The enzymes in the NAD(P)(H) cycle were selected to avoid cross-reactions, and the cascade was driven by irreversible fuel oxidation. As a proof-of-concept, formate oxidation was chosen as the fueling reaction. ATP regeneration was accomplished via the phosphorylation of NADH to NADPH and the subsequent transfer of the phosphate to ADP by a reversible NAD+ kinase. The cascade was able to regenerate ATP at a high rate (up to 0.74 mmol/L/h) for hours, and >90% conversion of ADP to ATP using monophosphate was also demonstrated. The cascade was used to regenerate ATP for use in cell free protein synthesis reactions, and the ATP production rate was further enhanced when powered by the multistep oxidation of methanol. The NAD(P)(H) cycle provides a simple cascade for the in vitro regeneration of ATP without the need for a pH-gradient or costly phosphate donors.
    Keywords:  Designed; membraneless NAD(P)(H) cycle enables continuous ATP regeneration to be driven by simple fuel oxidation
  34. BMC Complement Med Ther. 2023 Jun 27. 23(1): 212
      BACKGROUND: Cervical cancer (CC) is a common gynecological malignancy with high morbidity worldwide. Butyrate, a short-chain fatty acid produced by intestinal flora, has been reported to inhibit cervical carcinogenesis. This study aimed to investigate the pro-apoptotic effects of butyrate on CC and the underlying mechanisms.METHODS: Human HeLa and Ca Ski cells were used in this study. Cell proliferation, cell migration and invasion were detected by CCK-8 and EdU staining, transwell and wound healing assay, respectively. Cell cycle, mitochondrial membrane potential and apoptosis were evaluated by flow cytometry. Western blot and RT-qPCR were carried out to examine the related genes and proteins to the mitochondrial complex Ι and apoptosis. Metabolite changes were analyzed by energy metabolomics and assay kits. The association between G protein-coupled receptor 41, 43, 109a and CC prognosis was analyzed using data from The Cancer Genome Atlas (TCGA).
    RESULTS: CCK-8 results showed significant inhibition of CC cell proliferation induced by butyrate treatment, which was confirmed by EdU staining and cell cycle detection. Data from the transwell and wound healing assay revealed that CC cell migration was dramatically reduced following butyrate treatment. Additionally, invasiveness was also decreased by butyrate. Western blot analysis showed that cleaved Caspase 3 and cleaved PARP, the enforcers of apoptosis, were increased by butyrate treatment. The results of Annexin V/PI staining and TUNEL also showed an increase in butyrate-induced apoptotic cells. Expression of Cytochrome C (Cytc), Caspase 9, Bax, but not Caspase 12 or 8, were up-regulated under butyrate exposure. Mechanistically, the decrease in mitochondrial NADH and NAD + levels after treatment with butyrate was observed by energy metabolomics and the NAD+/NADH Assay Kit, similar to the effects of the complex Ι inhibitor rotenone. Western blot results also demonstrated that the constituent proteins of mitochondrial complex Ι were reduced by butyrate. Furthermore, mitochondria-dependent apoptosis has been shown to be initiated by inhibition of the complex Ι.
    CONCLUSION: Collectively, our results revealed that butyrate inhibited the proliferation, migration and invasion of CC cells, and induced mitochondrial-dependent apoptosis by inhibiting mitochondrial complex Ι.
    Keywords:  Apoptosis; Butyrate; Cervical cancer; Mitochondria; Mitochondrial complex Ι
  35. Cancer Commun (Lond). 2023 Jun 28.
      BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most lethal renal cancer. An overwhelming increase of patients experience tumor progression and unfavorable prognosis. However, the molecular events underlying ccRCC tumorigenesis and metastasis remain unclear. Therefore, uncovering the underlying mechanisms will pave the way for developing novel therapeutic targets for ccRCC. In this study, we sought to investigate the role of mitofusin-2 (MFN2) in supressing ccRCC tumorigenesis and metastasis.METHODS: The expression pattern and clinical significance of MFN2 in ccRCC were analyzed by using the Cancer Genome Atlas datasets and samples from our independent ccRCC cohort. Both in vitro and in vivo experiments, including cell proliferation, xenograft mouse models and transgenic mouse model, were used to determine the role of MFN2 in regulating the malignant behaviors of ccRCC. RNA-sequencing, mass spectrum analysis, co-immunoprecipitation, bio-layer interferometry and immunofluorescence were employed to elucidate the molecular mechanisms for the tumor-supressing role of MFN2.
    RESULTS: we reported a tumor-suppressing pathway in ccRCC, characterized by mitochondria-dependent inactivation of epidermal growth factor receptor (EGFR) signaling. This process was mediated by the outer mitochondrial membrane (OMM) protein MFN2. MFN2 was down-regulated in ccRCC and associated with favorable prognosis of ccRCC patients. in vivo and in vitro assays demonstrated that MFN2 inhibited ccRCC tumor growth and metastasis by suppressing the EGFR signaling pathway. In a kidney-specific knockout mouse model, loss of MFN2 led to EGFR pathway activation and malignant lesions in kidney. Mechanistically, MFN2 preferably binded small GTPase Rab21 in its GTP-loading form, which was colocalized with endocytosed EGFR in ccRCC cells. Through this EGFR-Rab21-MFN2 interaction, endocytosed EGFR was docked to mitochondria and subsequently dephosphorylated by the OMM-residing tyrosine-protein phosphatase receptor type J (PTPRJ).
    CONCLUSIONS: Our findings uncover an important non-canonical mitochondria-dependent pathway regulating EGFR signaling by the Rab21-MFN2-PTPRJ axis, which contributes to the development of novel therapeutic strategies for ccRCC.
    Keywords:  EGFR signaling pathway; MFN2; PTPRJ; Rab21; ccRCC