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Previous studies towards reduced oxygen availability have mostly focused on changes in total mRNA expression, neglecting underlying transcriptional and post-transcriptional events. Therefore, we generated a comprehensive overview of hypoxia-induced changes in total mRNA expression, global de novo transcription, and mRNA stability in monocytic THP-1 cells. Since hypoxic episodes often persist for prolonged periods, we further compared the adaptation to acute and chronic hypoxia. While total mRNA changes correlated well with enhanced transcription during short-term hypoxia, mRNA destabilization gained importance under chronic conditions. Reduced mRNA stability not only added to a compensatory attenuation of immune responses, but also, most notably, to the reduction in nuclear-encoded mRNAs associated with various mitochondrial functions. These changes may prevent the futile production of new mitochondria under conditions where mitochondria cannot exert their full metabolic function and are indeed actively removed by mitophagy. The post-transcriptional mode of regulation might further allow for the rapid recovery of mitochondrial capacities upon reoxygenation. Our results provide a comprehensive resource of functional mRNA expression dynamics and underlying transcriptional and post-transcriptional regulatory principles during the adaptation to hypoxia. Furthermore, we uncover that RNA stability regulation controls mitochondrial functions in the context of hypoxia.
Multiple myeloma (MM) is the second most common hematologic malignancy, which is characterized by clonal proliferation of neoplastic plasma cells in the bone marrow. This microenvironment is characterized by low oxygen levels (1–6% O2), known as hypoxia. For MM cells, hypoxia is a physiologic feature that has been described to promote an aggressive phenotype and to confer drug resistance. However, studies on hypoxia are scarce and show little conformity. Here, we analyzed the mRNA expression of previously determined hypoxia markers to define the temporal adaptation of MM cells to chronic hypoxia. Subsequent analyses of the global proteome in MM cells and the stromal cell line HS-5 revealed hypoxia-dependent regulation of proteins, which directly or indirectly upregulate glycolysis. In addition, chronic hypoxia led to MM-specific regulation of nine distinct proteins. One of these proteins is the cysteine protease legumain (LGMN), the depletion of which led to a significant growth disadvantage of MM cell lines that is enhanced under hypoxia. Thus, herein, we report a methodologic strategy to examine MM cells under physiologic hypoxic conditions in vitro and to decipher and study previously masked hypoxia-specific therapeutic targets such as the cysteine protease LGMN.
Tyrosine kinase inhibitors (TKIs) are currently the standard chemotherapeutic agents for the treatment of chronic myeloid leukemia (CML). However, due to TKI resistance acquisition in CML patients, identification of new vulnerabilities is urgently required for a sustained response to therapy. In this study, we have investigated metabolic reprogramming induced by TKIs independent of BCR-ABL1 alterations. Proteomics and metabolomics profiling of imatinib-resistant CML cells (ImaR) was performed. KU812 ImaR cells enhanced pentose phosphate pathway, glycogen synthesis, serine-glycine-one-carbon metabolism, proline synthesis and mitochondrial respiration compared with their respective syngeneic parental counterparts. Moreover, the fact that only 36% of the main carbon sources were utilized for mitochondrial respiration pointed to glycerol-phosphate shuttle as mainly contributors to mitochondrial respiration. In conclusion, CML cells that acquire TKIs resistance present a severe metabolic reprogramming associated with an increase in metabolic plasticity needed to overcome TKI-induced cell death. Moreover, this study unveils that KU812 Parental and ImaR cells viability can be targeted with metabolic inhibitors paving the way to propose novel and promising therapeutic opportunities to overcome TKI resistance in CML.
Background: Glucose metabolism in the tumor-microenvironment is a fundamental hallmark for tumor growth and intervention therein remains an attractive option for anti-tumor therapy. Whether tumor-derived factors such as microRNAs (miRs) regulate glucose metabolism in stromal cells, especially in tumor-associated macrophages (TAMs), to hijack them for trophic support, remains elusive.
Methods: Ago-RIP-Seq identified macrophage lactate dehydrogenase B (LDHB) as a target of tumor-derived miR-375 in both 2D/3D cocultures and in murine TAMs from a xenograft mouse model. The prognostic value was analyzed by ISH and multiplex IHC of breast cancer patient tissues. Functional consequences of the miR-375-LDHB axis in TAMs were investigated upon mimic/antagomir treatment by live metabolic flux assays, GC/MS, qPCR, Western blot, lentiviral knockdown and FACS. The therapeutic potential of a combinatorial miR-375-decoy/simvastatin treatment was validated by live cell imaging.
Results: Macrophage LDHB decreased in murine and human breast carcinoma. LDHB downregulation increase aerobic glycolysis and lactagenesis in TAMs in response to tumor-derived miR-375. Lactagenesis reduced fatty acid synthesis but activated SREBP2, which enhanced cholesterol biosynthesis in macrophages. LDHB downregulation skewed TAMs to function as a lactate and sterol/oxysterol source for the proliferation of tumor cells. Restoring of LDHB expression potentiated inhibitory effects of simvastatin on tumor cell proliferation.
Conclusion: Our findings identified a crucial role of LDHB in macrophages and established tumor-derived miR-375 as a novel regulator of macrophage metabolism in breast cancer, which might pave the way for strategies of combinatorial cancer cell/stroma cell interventions.
Hypoxia triggers several mechanisms to adapt cells to a low oxygen environment. Mitochondria are major consumers of oxygen and a potential source of reactive oxygen species (ROS). In response to hypoxia they exchange or modify distinct subunits of the respiratory chain and adjust their metabolism, especially lowering the citric acid cycle. Intermediates of the citric acid cycle participate in regulating hypoxia inducible factors (HIF), the key mediators of adaptation to hypoxia. Here we summarize how hypoxia conditions mitochondria with consequences for ROS-production and the HIF-pathway.
Human macrophages infiltrating hypoxic regions alter their metabolism, because oxygen becomes limited. Increased glycolysis is one of the most common cellular adaptations to hypoxia and mostly is regulated via hypoxia-inducible factor (HIF) and RAC-alpha serine/threonine–protein kinase (Akt) signaling, which gets activated under reduced oxygen content. We noticed that micro RNA (miR)-193a-3p enhances Akt phosphorylation at threonine 308 under hypoxia. In detail, miR-193a-3p suppresses the protein abundance of phosphatase PTC7 homolog (PPTC7), which in turn increases Akt phosphorylation. Lowering PPTC7 expression by siRNA or overexpressing miR-193a-3p increases Akt phosphorylation. Vice versa, inhibition of miR-193a-3p attenuates Akt activation and prevents a subsequent increase of glycolysis under hypoxia. Excluding effects of miR-193a-3p and Akt on HIF expression, stabilization, and function, we noticed phosphorylation of 6 phosphofructo-2-kinase/fructose 2,6-bisphosphatase PFKFB3 in response to the PI3K/Akt/mTOR signaling cascade. Inhibition of PFKFB3 blocked an increased glycolytic flux under hypoxia. Apparently, miR-193a-3p balances Akt phosphorylation and dephosphorylation by affecting PPTC7 protein amount. Suppression of PPTC7 increases Akt activation and phosphorylation of PFKFB3, which culminates in higher rates of glycolysis under hypoxia.
Ferroptosis is an iron-dependent form of cell death, which is triggered by disturbed membrane integrity due to an overproduction of lipid peroxides. Induction of ferroptosis comprises several alterations, i.e. altered iron metabolism, response to oxidative stress, or lipid peroxide production. At the physiological level transcription, translation, and microRNAs add to the appearance and/or activity of building blocks that negatively or positively balance ferroptosis. Ferroptosis contributes to tissue damage in the case of, e.g., brain and heart injury but may be desirable to overcome chemotherapy resistance. For a more complete picture, it is crucial to also consider the cellular microenvironment, which during inflammation and in the tumor context is dominated by hypoxia. This graphical review visualizes basic mechanisms of ferroptosis, categorizes general inducers and inhibitors of ferroptosis, and puts a focus on microRNAs, iron homeostasis, and hypoxia as regulatory components.
Hypoxia inhibits ferritinophagy, increases mitochondrial ferritin, and protects from ferroptosis
(2020)
Highlights
• Hypoxia decreases NCOA4 transcription in primary human macrophages.
• NCOA4 mRNA is a target of miR-6862-5p.
• Lowering NCOA4 increases FTMT abundance under hypoxia.
• FTMT and FTH protect from ferroptosis.
• Tumor cells lack the hypoxic decrease of NCOA4 and fail to stabilize FTMT.
Abstract
Cellular iron, at the physiological level, is essential to maintain several metabolic pathways, while an excess of free iron may cause oxidative damage and/or provoke cell death. Consequently, iron homeostasis has to be tightly controlled. Under hypoxia these regulatory mechanisms for human macrophages are not well understood. Hypoxic primary human macrophages reduced intracellular free iron and increased ferritin expression, including mitochondrial ferritin (FTMT), to store iron. In parallel, nuclear receptor coactivator 4 (NCOA4), a master regulator of ferritinophagy, decreased and was proven to directly regulate FTMT expression. Reduced NCOA4 expression resulted from a lower rate of hypoxic NCOA4 transcription combined with a micro RNA 6862-5p-dependent degradation of NCOA4 mRNA, the latter being regulated by c-jun N-terminal kinase (JNK). Pharmacological inhibition of JNK under hypoxia increased NCOA4 and prevented FTMT induction. FTMT and ferritin heavy chain (FTH) cooperated to protect macrophages from RSL-3-induced ferroptosis under hypoxia as this form of cell death is linked to iron metabolism. In contrast, in HT1080 fibrosarcome cells, which are sensitive to ferroptosis, NCOA4 and FTMT are not regulated. Our study helps to understand mechanisms of hypoxic FTMT regulation and to link ferritinophagy and macrophage sensitivity to ferroptosis.
Hypoxia poses a stress to cells and decreases mitochondrial respiration, in part by electron transport chain (ETC) complex reorganization. While metabolism under acute hypoxia is well characterized, alterations under chronic hypoxia largely remain unexplored. We followed oxygen consumption rates in THP-1 monocytes during acute (16 h) and chronic (72 h) hypoxia, compared to normoxia, to analyze the electron flows associated with glycolysis, glutamine, and fatty acid oxidation. Oxygen consumption under acute hypoxia predominantly demanded pyruvate, while under chronic hypoxia, fatty acid- and glutamine-oxidation dominated. Chronic hypoxia also elevated electron-transferring flavoproteins (ETF), and the knockdown of ETF–ubiquinone oxidoreductase lowered mitochondrial respiration under chronic hypoxia. Metabolomics revealed an increase in citrate under chronic hypoxia, which implied glutamine processing to α-ketoglutarate and citrate. Expression regulation of enzymes involved in this metabolic shunting corroborated this assumption. Moreover, the expression of acetyl-CoA carboxylase 1 increased, thus pointing to fatty acid synthesis under chronic hypoxia. Cells lacking complex I, which experienced a markedly impaired respiration under normoxia, also shifted their metabolism to fatty acid-dependent synthesis and usage. Taken together, we provide evidence that chronic hypoxia fuels the ETC via ETFs, increasing fatty acid production and consumption via the glutamine-citrate-fatty acid axis.
Mitochondrial derived reactive oxygen species (mtROS) are known for their signaling qualities in both physiology and pathology. To elucidate mitochondrial complex I-dependent ROS-signaling after lipopolysaccharide (LPS)-stimulation THP-1 macrophages with a knockdown of the transmembrane protein TMEM126B were generated. TMEM knockdown cells (sh126B) showed a reduced assembly of complex I and attenuated mtROS production. In these cells we identified protein oxidization by mtROS upon LPS-treatment using the BIAM switch assay coupled to liquid chromatography and mass spectrometry. One of the identified targets of mtROS was succinate dehydrogenase (SDH) flavoprotein subunit A (SDHA). Oxidation of SDHA decreased its enzymatic activity and pharmacological inhibition of SDH in turn stabilized hypoxia inducible factor (HIF)-1α and caused the subsequent, sustained expression of interleukin-1β (IL-1β). Oxidation of SDHA in sh126B cells was attenuated, while pharmacological inhibition of SDH by atpenin A5 restored IL-1β expression in sh126B cells upon LPS-treatment. Conclusively, oxidation of SDH by mtROS links an altered metabolism, i.e. succinate accumulation to HIF-1-driven, inflammatory changes in macrophages.