ABSTRACT
BACKGROUND: The aberrant expression of phosphofructokinase-platelet (PFKP) plays a crucial role in the development of various human cancers by modifying diverse biological functions. However, the precise molecular mechanisms underlying the role of PFKP in head and neck squamous cell carcinoma (HNSCC) are not fully elucidated. METHODS: We assessed the expression levels of PFKP and c-Myc in tumor and adjacent normal tissues from 120 HNSCC patients. A series of in vitro and in vivo experiments were performed to explore the impact of the feedback loop between PFKP and c-Myc on HNSCC progression. Additionally, we explored the therapeutic effects of targeting PFKP and c-Myc in HNSCC using Patient-Derived Organoids (PDO), Cell Line-Derived Xenografts, and Patients-Derived Xenografts. RESULTS: Our findings indicated that PFKP is frequently upregulated in HNSCC tissues and cell lines, correlating with poor prognosis. Our in vitro and in vivo experiments demonstrate that elevated PFKP facilitates cell proliferation, angiogenesis, and metastasis in HNSCC. Mechanistically, PFKP increases the ERK-mediated stability of c-Myc, thereby driving progression of HNSCC. Moreover, c-Myc stimulates PFKP expression at the transcriptional level, thus forming a positive feedback loop between PFKP and c-Myc. Additionally, our multiple models demonstrate that co-targeting PFKP and c-Myc triggers synergistic anti-tumor effects in HNSCC. CONCLUSION: Our study demonstrates the critical role of the PFKP/c-Myc positive feedback loop in driving HNSCC progression and suggests that simultaneously targeting PFKP and c-Myc may be a novel and effective therapeutic strategy for HNSCC.
Subject(s)
Disease Progression , Feedback, Physiological , Gene Expression Regulation, Neoplastic , Head and Neck Neoplasms , Proto-Oncogene Proteins c-myc , Squamous Cell Carcinoma of Head and Neck , Humans , Squamous Cell Carcinoma of Head and Neck/metabolism , Squamous Cell Carcinoma of Head and Neck/pathology , Squamous Cell Carcinoma of Head and Neck/genetics , Proto-Oncogene Proteins c-myc/metabolism , Proto-Oncogene Proteins c-myc/genetics , Animals , Mice , Head and Neck Neoplasms/metabolism , Head and Neck Neoplasms/pathology , Head and Neck Neoplasms/genetics , Cell Line, Tumor , Phosphofructokinase-1, Type C/metabolism , Phosphofructokinase-1, Type C/genetics , Cell Proliferation , Prognosis , Female , Male , Xenograft Model Antitumor Assays , Biomarkers, Tumor/metabolismABSTRACT
Aerobic glycolysis is critical for cancer progression and can be exploited in cancer therapy. Here, we report that the human carboxymethylenebutenolidase homolog (carboxymethylenebutenolidase-like [CMBL]) acts as a tumor suppressor by reprogramming glycolysis in colorectal cancer (CRC). The anti-cancer action of CMBL is mediated through its interactions with the E3 ubiquitin ligase TRIM25 and the glycolytic enzyme phosphofructokinase-1 platelet type (PFKP). Ectopic CMBL enhances TRIM25 binding to PFKP, leading to the ubiquitination and proteasomal degradation of PFKP. Interestingly, CMBL is transcriptionally activated by p53 in response to genotoxic stress, and p53 activation represses glycolysis by promoting PFKP degradation. Remarkably, CMBL deficiency, which impairs p53's ability to inhibit glycolysis, makes tumors more sensitive to a combination therapy involving the glycolysis inhibitor 2-deoxyglucose. Taken together, our study demonstrates that CMBL suppresses CRC growth by inhibiting glycolysis and suggests a potential combination strategy for the treatment of CMBL-deficient CRC.
Subject(s)
Neoplasms , Phosphofructokinase-1, Type C , Humans , Cell Line, Tumor , Glucose/metabolism , Glycolysis , Phosphofructokinase-1/metabolism , Phosphofructokinase-1, Type C/metabolism , Phosphofructokinases/metabolism , Tumor Suppressor Protein p53/metabolismABSTRACT
Purpose Cutaneous melanoma is an aggressive and deadly cancer resulting from malignant transformation of cells involved in skin pigmentation. Glycolysis is widely implicated in cancer progression, but its precise role in melanoma has not been extensively studied. Here, we investigated the role of the glycolysis regulator phosphofructokinase 1 platelet isoform (PFKP) in melanoma progression. Methods PFKP expression in human melanoma tissues was analyzed by immunohistochemistry. Knockdown of PFKP by siRNA and overexpression of PFKP were performed to evaluate its functions in vitro. CCK-8 assay was used to assess cell proliferation. Glycolytic activity was determined via measurement of extracellular acidification rate (ECAR), lactic acid level, and ATP content. A tumor xenograft model was used to test the function of PFKP in vivo. Results PFKP upregulation was observed in human melanoma tissues and correlated with poor patient survival. Knockdown of PFKP in human melanoma cells suppressed cell proliferation and reduced ECAR, ATP levels, and lactic acid levels, while overexpression of PFKP displayed the opposite effects. In vivo, knockdown of PFKP in melanoma cells markedly reduced tumorigenesis. Inhibitory effects on cell proliferation, glycolysis, and tumorigenesis due to PFKP knockdown were further augmented upon treatment with the glycolysis inhibitor 2-deoxy-D-glucose (2-DG). Conclusion Collectively, these results indicate that PFKP expression in melanoma cells increases proliferation and glycolytic activity in vitro and promotes tumorigenesis in vivo, suggesting that suppression of PKFP and inhibition of glycolysis may potently suppress melanoma progression (AU)
Subject(s)
Humans , Phosphofructokinase-1, Type C/genetics , Phosphofructokinase-1, Type C/metabolism , Skin Neoplasms/genetics , Skin Neoplasms/metabolism , Melanoma/genetics , Melanoma/metabolism , Adenosine Triphosphate/metabolism , Cell Proliferation , Carcinogenesis , Cell Line, Tumor , Glycolysis/genetics , Lactic Acid/metabolismABSTRACT
PURPOSE: Cutaneous melanoma is an aggressive and deadly cancer resulting from malignant transformation of cells involved in skin pigmentation. Glycolysis is widely implicated in cancer progression, but its precise role in melanoma has not been extensively studied. Here, we investigated the role of the glycolysis regulator phosphofructokinase 1 platelet isoform (PFKP) in melanoma progression. METHODS: PFKP expression in human melanoma tissues was analyzed by immunohistochemistry. Knockdown of PFKP by siRNA and overexpression of PFKP were performed to evaluate its functions in vitro. CCK-8 assay was used to assess cell proliferation. Glycolytic activity was determined via measurement of extracellular acidification rate (ECAR), lactic acid level, and ATP content. A tumor xenograft model was used to test the function of PFKP in vivo. RESULTS: PFKP upregulation was observed in human melanoma tissues and correlated with poor patient survival. Knockdown of PFKP in human melanoma cells suppressed cell proliferation and reduced ECAR, ATP levels, and lactic acid levels, while overexpression of PFKP displayed the opposite effects. In vivo, knockdown of PFKP in melanoma cells markedly reduced tumorigenesis. Inhibitory effects on cell proliferation, glycolysis, and tumorigenesis due to PFKP knockdown were further augmented upon treatment with the glycolysis inhibitor 2-deoxy-D-glucose (2-DG). CONCLUSION: Collectively, these results indicate that PFKP expression in melanoma cells increases proliferation and glycolytic activity in vitro and promotes tumorigenesis in vivo, suggesting that suppression of PKFP and inhibition of glycolysis may potently suppress melanoma progression.
Subject(s)
Melanoma , Phosphofructokinase-1, Type C , Skin Neoplasms , Humans , Adenosine Triphosphate/metabolism , Carcinogenesis , Cell Line, Tumor , Cell Proliferation/genetics , Glycolysis/genetics , Lactic Acid/metabolism , Phosphofructokinase-1, Type C/genetics , Phosphofructokinase-1, Type C/metabolismABSTRACT
BACKGROUND: Overexpression of PD-L1 is observed in many types of human cancer, including glioblastoma (GBM) and contributes to tumor immune evasion. In addition, GBM shows highly-activated aerobic glycolysis due to overexpression of phosphofructokinase 1 platelet isoform (PFKP), which the key enzyme in the glycolysis. However, it remains unclear whether the metabolic enzyme PFKP plays a role in the regulation of PD-L1 expression and GBM immune evasion. OBJECTIVE: We aimed to investigate the non-metabolic role of PFKP in PD-L1 expression-induced GBM immune evasion. METHODS: The mechanisms of PFKP-induced PD-L1 expression were studied by several experiments, including real-time PCR, immunoblot analysis, and ATP production. The coculture experiments using GBM cell and T cells were performed to evaluate the effect of PFKP on T cell activation. The clinical relationship between PFKP and PD-L1 was analyzed in The Cancer Genome Atlas (TCGA) database and in human GBM specimens. RESULTS: We showed that PFKP promotes EGFR activation-induced PD-L1 expression in human GBM cells. Importantly, we demonstrated that EGFR-phosphorylated PFKP Y64 plays an important role in AKT-mediated ß-catenin transactivation and subsequent PD-L1 transcriptional expression, thereby enhancing the GBM immune evasion. In addition, based on our findings, the levels of PFKP Y64 phosphorylation are positively correlated with PD-L1 expression in human GBM specimens, highlighting the clinical significance of PFKP Y64 phosphorylation in the GBM immune evasion. CONCLUSION: These findings provide new mechanistic insight into the regulation of PD-L1 expression by a non-metabolic function of PFKP on tumor cells.
Subject(s)
Glioblastoma , Phosphofructokinase-1, Type C , Humans , B7-H1 Antigen/genetics , B7-H1 Antigen/metabolism , ErbB Receptors/metabolism , Glioblastoma/genetics , Glioblastoma/metabolism , Glioblastoma/pathology , Immune Evasion , Phosphorylation , Protein Isoforms/metabolism , Phosphofructokinase-1, Type C/genetics , Phosphofructokinase-1, Type C/metabolismABSTRACT
Fbxo7 is associated with cancer and Parkinson's disease. Although Fbxo7 recruits substrates for SCF-type ubiquitin ligases, it also promotes Cdk6 activation in a ligase-independent fashion. We discovered PFKP, the gatekeeper of glycolysis, in a screen for Fbxo7 substrates. PFKP is an essential Cdk6 substrate in some T-ALL cells. We investigated the molecular relationship between Fbxo7, Cdk6, and PFKP, and the effect of Fbxo7 on T cell metabolism, viability, and activation. Fbxo7 promotes Cdk6-independent ubiquitination and Cdk6-dependent phosphorylation of PFKP. Importantly, Fbxo7-deficient cells have reduced Cdk6 activity, and hematopoietic and lymphocytic cells show high expression and significant dependency on Fbxo7. CD4+ T cells with reduced Fbxo7 show increased glycolysis, despite lower cell viability and activation levels. Metabolomic studies of activated CD4+ T cells confirm increased glycolytic flux in Fbxo7-deficient cells, alongside altered nucleotide biosynthesis and arginine metabolism. We show Fbxo7 expression is glucose-responsive at the mRNA and protein level and propose Fbxo7 inhibits PFKP and glycolysis via its activation of Cdk6.
Subject(s)
Cyclin-Dependent Kinase 6 , F-Box Proteins , Phosphofructokinase-1, Type C , T-Lymphocytes , Cyclin-Dependent Kinase 6/metabolism , F-Box Proteins/genetics , F-Box Proteins/metabolism , Glycolysis , Humans , Phosphofructokinase-1, Type C/metabolism , T-Lymphocytes/metabolism , UbiquitinationABSTRACT
Alcohol abuse, reported by 1/8th critically ill patients, is an independent risk factor for death in sepsis. Sepsis kills over 270,000 patients/year in the US. We reported that the ethanol-exposure suppresses innate-immune response, pathogen clearance, and decreases survival in sepsis-mice via sirtuin 2 (SIRT2). SIRT2 is an NAD+-dependent histone-deacetylase with anti-inflammatory properties. We hypothesized that in ethanol-exposed macrophages, SIRT2 suppresses phagocytosis and pathogen clearance by regulating glycolysis. Immune cells use glycolysis to fuel increased metabolic and energy demand of phagocytosis. Using ethanol-exposed mouse bone marrow- and human blood monocyte-derived macrophages, we found that SIRT2 mutes glycolysis via deacetylating key glycolysis regulating enzyme phosphofructokinase-platelet isoform (PFKP), at mouse lysine 394 (mK394, human: hK395). Acetylation of PFKP at mK394 (hK395) is crucial for PFKP function as a glycolysis regulating enzyme. The PFKP also facilitates phosphorylation and activation of autophagy related protein 4B (Atg4B). Atg4B activates microtubule associated protein 1 light chain-3B (LC3). LC3 is a driver of a subset of phagocytosis, the LC3-associated phagocytosis (LAP), which is crucial for segregation and enhanced clearance of pathogens, in sepsis. We found that in ethanol-exposed cells, the SIRT2-PFKP interaction leads to decreased Atg4B-phosphorylation, decreased LC3 activation, repressed phagocytosis and LAP. Genetic deficiency or pharmacological inhibition of SIRT2 reverse PFKP-deacetylation, suppressed LC3-activation and phagocytosis including LAP, in ethanol-exposed macrophages to improve bacterial clearance and survival in ethanol with sepsis mice.
Subject(s)
Ethanol , Macrophages , Phagocytosis , Phosphofructokinase-1, Type C , Sirtuin 2 , Animals , Humans , Mice , Ethanol/adverse effects , Macrophages/drug effects , Sirtuin 2/metabolism , Phosphofructokinase-1, Type C/metabolismABSTRACT
RNA editing is a feature of RNA maturation resulting in the formation of transcripts whose sequence differs from the genome template. Brain RNA editing may be altered in Alzheimer's disease (AD). Here, we analyzed data from 1,865 brain samples covering 9 brain regions from 1,074 unrelated subjects on a transcriptome-wide scale to identify inter-regional differences in RNA editing. We expand the list of known brain editing events by identifying 58,761 previously unreported events. We note that only a small proportion of these editing events are found at the protein level in our proteome-wide validation effort. We also identified the occurrence of editing events associated with AD dementia, neuropathological measures and longitudinal cognitive decline in: SYT11, MCUR1, SOD2, ORAI2, HSDL2, PFKP, and GPRC5B. Thus, we present an extended reference set of brain RNA editing events, identify a subset that are found to be expressed at the protein level, and extend the narrative of transcriptomic perturbation in AD to RNA editing.
Subject(s)
Alzheimer Disease/genetics , ORAI2 Protein/genetics , RNA Editing , RNA/genetics , Synaptotagmins/genetics , Transcriptome , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Atlases as Topic , Brain/metabolism , Brain/pathology , Brain Chemistry , Gene Expression Profiling , Humans , Hydroxysteroid Dehydrogenases/genetics , Hydroxysteroid Dehydrogenases/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , ORAI2 Protein/metabolism , Phosphofructokinase-1, Type C/genetics , Phosphofructokinase-1, Type C/metabolism , RNA/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Synaptotagmins/metabolismABSTRACT
BACKGROUND: Glycolysis reprogramming is deeply involved in the progression of hepatocellular carcinoma (HCC), in which HCC cells with stemness traits play important roles as well. Thus, whether platelet isoform of phosphofructokinase 1 (PFKP), a rate-limiting enzyme in glycolysis, contributes to the maintenance of stemness of HCC cells is worth investigation. METHODS: PFKP levels were compared between human hepatocellular carcinoma and adjacent normal tissues by Western blotting and immunohistochemistry. The relationship between PFKP expression and clinic pathological features was also analyzed. Furthermore, the colony formation capabilities and the levels of stemness markers (ALDH1, CD44, CD133, Sox-2) as well as ß-catenin were compared between HCC cells either undergoing PFKP silencing or overexpression. RESULTS: PFKP levels were higher in HCC as compared to normal hepatic tissues. Silencing PFKP decreased HCC proliferation, colony formation capabilities, and levels of stemness markers and ß-catenin; whereas overexpressing PFKP demonstrated the opposite effects. CONCLUSION: PFKP promoted HCC proliferation and contributed to the maintenance of HCC stemness. Silencing PFKP could restrain the stemness of HCC, suggesting that PFKP may be a potential therapeutic target for HCC treatment.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/metabolism , Neoplastic Stem Cells/metabolism , Phosphofructokinase-1, Type C/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Proliferation/physiology , Glycolysis/physiology , Humans , Liver Neoplasms/pathologyABSTRACT
Phosphofructokinase 1 (PFK1) is expressed in T cell acute lymphoblastic leukemia (T-ALL), where its upregulation is linked with cancer progression. While PFK1 functions in the glycolysis pathway within the cytoplasm, it is also present in the nucleus where it regulates gene transcription. In this issue of the JCI, Xueliang Gao, Shenghui Qin, et al. focus their mechanism-based investigation on the nucleocytoplasmic shuttling aspect of the PFK1 platelet isoform, PFKP. Functional nuclear export and localization sequences stimulated CXC chemokine receptor type 4 (CXCR4) expression to promote T-ALL invasion that involved cyclin D3/CDK6, c-Myc, and importin-9. Since the presence of nuclear PFKP is associated with poor survival in T-ALL, nuclear PFKP-induced CXCR4 expression might serve as a prognostic marker for T-ALL. More promising, though, are the mechanistic insights suggesting that approaches to dampening metastatic migration may have application to benefit patients with T-ALL.
Subject(s)
Phosphofructokinase-1, Type C , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Glycolysis , Humans , Phosphofructokinase-1, Type C/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Prognosis , Receptors, CXCR4/genetics , T-Lymphocytes/metabolismABSTRACT
PFKP (phosphofructokinase, platelet), the major isoform of PFK1 expressed in T cell acute lymphoblastic leukemia (T-ALL), is predominantly expressed in the cytoplasm to carry out its glycolytic function. Our study showed that PFKP is a nucleocytoplasmic shuttling protein with functional nuclear export and nuclear localization sequences (NLSs). Cyclin D3/CDK6 facilitated PFKP nuclear translocation by dimerization and by exposing the NLS of PFKP to induce the interaction between PFKP and importin 9. Nuclear PFKP stimulated the expression of C-X-C chemokine receptor type 4 (CXCR4), a chemokine receptor regulating leukemia homing/infiltration, to promote T-ALL cell invasion, which depended on the activity of c-Myc. In vivo experiments showed that nuclear PFKP promoted leukemia homing/infiltration into the bone marrow, spleen, and liver, which could be blocked with CXCR4 antagonists. Immunohistochemical staining of tissues from a clinically well-annotated cohort of T cell lymphoma/leukemia patients showed nuclear PFKP localization in invasive cancers, but not in nonmalignant T lymph node or reactive hyperplasia. The presence of nuclear PFKP in these specimens correlated with poor survival in patients with T cell malignancy, suggesting the potential utility of nuclear PFKP as a diagnostic marker.
Subject(s)
Phosphofructokinase-1, Type C/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Receptors, CXCR4/metabolism , Active Transport, Cell Nucleus , Animals , Biomarkers, Tumor/metabolism , Cyclin-Dependent Kinase 6/metabolism , Female , Humans , Karyopherins/metabolism , Lymphocytes, Tumor-Infiltrating/metabolism , Lymphocytes, Tumor-Infiltrating/pathology , Male , Mice , Mice, Inbred NOD , Mice, SCID , Middle Aged , Models, Molecular , Neoplasm Invasiveness/pathology , Neoplasm Invasiveness/physiopathology , Phosphofructokinase-1, Type C/chemistry , Phosphofructokinase-1, Type C/genetics , Prognosis , Protein Interaction Domains and Motifs , Proto-Oncogene Proteins c-myc/metabolism , Tumor Cells, CulturedABSTRACT
Triptolide (TP), a primary bioactive ingredient isolated from the traditional Chinese herbal medicine Tripterygium wilfordii Hook. F. (TWHF), has attracted great interest for its therapeutic biological activities in inflammation and autoimmune disease. However, its clinical use is limited by severe testicular toxicity, and the underlying mechanism has not been elucidated. Our preliminary evidence demonstrated that TP disrupted glucose metabolism and caused testicular toxicity. During spermatogenesis, Sertoli cells (SCs) provide lactate as an energy source to germ cells by glycolysis. The transcription factors GATA-binding protein 4 (GATA4) and specificity protein 1 (Sp1) can regulate glycolysis. Based on this evidence, we speculate that TP causes abnormal glycolysis in SCs by influencing the expression of the transcription factors GATA4 and Sp1. The mechanism of TP-induced testicular toxicity was investigated in vitro and in vivo. The data indicated that TP decreased glucose consumption, lactate production, and the mRNA levels of glycolysis-related transporters and enzymes. TP also downregulated the protein expression of the transcription factors GATA4 and Sp1, as well as the glycolytic enzyme phosphofructokinase platelet (PFKP). Phosphorylated GATA4 and nuclear GATA4 protein levels were reduced in a dose- and time-dependent manner after TP incubation. Similar effects were observed in shGata4-treated TM4 cells and BALB/c mice administered 0.4 mg/kg TP for 28 days, and glycolysis was also inhibited. Gata4 knockdown downregulated Sp1 and PFKP expression. Furthermore, the Sp1 inhibitor plicamycin inhibited PFKP protein levels in TM4 cells. In conclusion, TP inhibited GATA4-mediated glycolysis by suppressing Sp1-dependent PFKP expression in SCs and caused testicular toxicity.
Subject(s)
Diterpenes/pharmacology , GATA4 Transcription Factor/metabolism , Glycolysis/drug effects , Phenanthrenes/pharmacology , Phosphofructokinase-1, Type C/metabolism , Sertoli Cells/drug effects , Sp1 Transcription Factor/metabolism , Animals , Cell Line , Cell Proliferation , Cell Survival/drug effects , Down-Regulation , Epoxy Compounds/pharmacology , GATA4 Transcription Factor/drug effects , GATA4 Transcription Factor/genetics , Gene Expression Regulation/drug effects , HEK293 Cells , Humans , Male , Mice , Mice, Inbred ICR , Phosphofructokinase-1, Type C/drug effects , Phosphofructokinase-1, Type C/genetics , Sertoli Cells/metabolism , Signal Transduction/drug effects , Sp1 Transcription Factor/drug effects , Sp1 Transcription Factor/geneticsABSTRACT
ATG4B facilitates autophagy by promoting autophagosome maturation through the reversible lipidation and delipidation of LC3. Recent reports have shown that phosphorylation of ATG4B regulates its activity and LC3 processing, leading to modulate autophagy activity. However, the mechanism about how ATG4B phosphorylation is involved in amino acid deprivation-induced autophagy is unclear. Here, we combined the tandem affinity purification with mass spectrometry (MS) and identified the ATG4B-interacting proteins including its well-known partner gamma-aminobutyric acid receptor-associated protein (GABARAP, a homolog of LC3) and phosphofructokinase 1 platelet isoform (PFKP). Further immunoprecipitation assays showed that amino acid deprivation strengthened the interaction between ATG4B and PFKP. By genetic depletion of PFKP using CRISPR/Cas9, we uncovered that PFKP loss reduced the degradation of LC3-II and p62 due to a partial block in autophagic flux. Furthermore, MS analysis of Flag-tagged ATG4B immunoprecipitates identified phosphorylation of ATG4B serine 34 residue (S34) and PFKP serine 386 residue (S386) under amino acid deprivation condition. In vitro kinase assay validated that PFKP functioning as a protein kinase phosphorylated ATG4B at S34. This phosphorylation could enhance ATG4B activity and p62 degradation. In addition, PFKP S386 phosphorylation was important to ATG4B S34 phosphorylation and autophagy in HEK293T cells. In brief, our findings describe that PFKP, a rate-limiting enzyme in the glycolytic pathway, functions as a protein kinase for ATG4B to regulate ATG4B activity and autophagy under amino acid deprivation condition.
Subject(s)
Autophagy-Related Proteins/metabolism , Cysteine Endopeptidases/metabolism , Phosphofructokinase-1, Type C/metabolism , Autophagy , HEK293 Cells , Humans , PhosphorylationABSTRACT
BACKGROUND: Our previous studies have shown that the E3 ubiquitin ligase of HMG-CoA reductase degradation 1 (HRD1) functions as a tumor suppressor, as overexpression of HRD1 suppressed breast cancer proliferation and invasion. However, its role in breast cancer cell glucose metabolism was unclear. Here, our aim was to uncover the role and molecular mechanisms of HRD1 in regulating aerobic glycolysis in breast cancer. METHODS: The effect of HRD1 on robic glycolysis in breast cancer cells were assessed. Then the proliferation, colony formation ability, invasion and migration of breast cancer cells were evaluated. The relationship between HRD1 and PFKP was validated by Mass spectrometry analysis, immunofluorescence and co-immunoprecipitation. The level of PFKP ubiquitination was measured using ubiquitylation assay. Furthermore, the tumor growth and metastasis in mice xenografts were observed. RESULTS: We found that upregulation of HRD1 clearly decreased aerobic glycolysis, and subsequently inhibited breast cancer proliferation and invasion. Mass spectrometry analysis results revealed a large HRD1 interactome, which included PFKP (platelet isoform of phosphofructokinase), a critical enzyme involved in the Warburg Effect in breast cancer. Mechanistically, HRD1 interacted and colocalized with PFKP in the cytoplasm, targeted PFKP for ubiquitination and degradation, and ultimately reduced PFKP expression and activity in breast cancer cells. HRD1 inhibited breast cancer growth and metastasis in vivo through a PFKP-dependent way CONCLUSIONS: Our findings reveal a new regulatory role of HRD1 in Warburg effect and provide a key contributor in breast cancer metabolism. Video abstract.
Subject(s)
Breast Neoplasms/metabolism , Mammary Neoplasms, Experimental/metabolism , Phosphofructokinase-1, Type C/metabolism , Ubiquitin-Protein Ligases/metabolism , Adenosine Triphosphate/metabolism , Animals , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line , Cell Movement , Cell Proliferation , Disease Progression , Female , Glucose/metabolism , Glycolysis , Humans , Lactic Acid/metabolism , Lung Neoplasms/metabolism , Lung Neoplasms/secondary , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/pathology , Mice, Inbred BALB C , Mice, Nude , Phosphofructokinase-1, Type C/genetics , Signal Transduction , Ubiquitin-Protein Ligases/genetics , Up-RegulationABSTRACT
R-2-hydroxyglutarate (R-2HG), a metabolite produced by mutant isocitrate dehydrogenases (IDHs), was recently reported to exhibit anti-tumor activity. However, its effect on cancer metabolism remains largely elusive. Here we show that R-2HG effectively attenuates aerobic glycolysis, a hallmark of cancer metabolism, in (R-2HG-sensitive) leukemia cells. Mechanistically, R-2HG abrogates fat-mass- and obesity-associated protein (FTO)/N6-methyladenosine (m6A)/YTH N6-methyladenosine RNA binding protein 2 (YTHDF2)-mediated post-transcriptional upregulation of phosphofructokinase platelet (PFKP) and lactate dehydrogenase B (LDHB) (two critical glycolytic genes) expression and thereby suppresses aerobic glycolysis. Knockdown of FTO, PFKP, or LDHB recapitulates R-2HG-induced glycolytic inhibition in (R-2HG-sensitive) leukemia cells, but not in normal CD34+ hematopoietic stem/progenitor cells, and inhibits leukemogenesis in vivo; conversely, their overexpression reverses R-2HG-induced effects. R-2HG also suppresses glycolysis and downregulates FTO/PFKP/LDHB expression in human primary IDH-wild-type acute myeloid leukemia (AML) cells, demonstrating the clinical relevance. Collectively, our study reveals previously unrecognized effects of R-2HG and RNA modification on aerobic glycolysis in leukemia, highlighting the therapeutic potential of targeting cancer epitranscriptomics and metabolism.
Subject(s)
Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics , Antineoplastic Agents/pharmacology , Glutarates/pharmacology , Glycolysis/genetics , Lactate Dehydrogenases/genetics , Leukemia, Myeloid, Acute/drug therapy , Phosphofructokinase-1, Type C/genetics , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/antagonists & inhibitors , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/metabolism , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Fluorouracil/pharmacology , Gene Expression Regulation, Neoplastic , Glycolysis/drug effects , HEK293 Cells , Humans , K562 Cells , Lactate Dehydrogenases/antagonists & inhibitors , Lactate Dehydrogenases/metabolism , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/mortality , Leukemia, Myeloid, Acute/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Oxidative Phosphorylation/drug effects , Phosphofructokinase-1, Type C/antagonists & inhibitors , Phosphofructokinase-1, Type C/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Signal Transduction , Survival Analysis , Xenograft Model Antitumor AssaysABSTRACT
Background: Glycolysis dysfunction is an important pathogenesis of podocyte injury in diabetic kidney disease (DKD). Foot process fusion of podocytes and increased albuminuria are markers of early DKD. Moreover, cytoskeletal remodeling has been found to be involved in the foot process fusion of podocytes. However, the connections between cytoskeletal remodeling and alterations of glycolysis in podocytes in DKD have not been clarified. Methods: mRNA sequencing of glomeruli obtained from db/db and db/m mice with albuminuria was performed to analyze the expression profiling of genes in glucose metabolism. Expressions of phosphofructokinase platelet type (PFKP) in the glomeruli of DKD patients were detected. Clotrimazole (CTZ) was used to explore the renal effects of PFKP inhibition in diabetic mice. Using Pfkp siRNA or recombinant plasmid to manipulate PFKP expression, the effects of PFKP on high glucose (HG) induced podocyte damage were assessed in vitro. The levels of fructose-1,6-bisphosphate (FBP) were measured. Targeted metabolomics was performed to observe the alterations of the metabolites in glucose metabolism after HG stimulation. Furthermore, aldolase type b (Aldob) siRNA or recombinant plasmid were applied to evaluate the influence of FBP level alteration on podocytes. FBP was directly added to podocyte culture media. Db/db mice were treated with FBP to investigate its effects on their kidney. Results: mRNA sequencing showed that glycolysis enzyme genes were altered, characterized by upregulation of upstream genes (Hk1, and Pfkp) and down-regulation of downstream genes of glycolysis (Pkm, and Ldha). Moreover, the expression of PFKP was increased in glomeruli of DKD patients. The CTZ group presented more severe renal damage. In vitro, the Pfkp siRNA group and ALDOB overexpression group showed much more induced cytoskeletal remodeling in podocytes, while overexpression of PFKP and suppression of ALDOB in vitro rescued podocytes from cytoskeletal remodeling through regulation of FBP levels and inhibition of the RhoA/ROCK1 pathway. Furthermore, targeted metabolomics showed FBP level was significantly increased in HG group compared with the control group. Exogenous FBP addition reduced podocyte cytoskeletal remodeling and renal damage of db/db mice. Conclusions: These findings provide evidence that PFKP may be a potential target for podocyte injury in DN and provide a rationale for applying podocyte glycolysis enhancing agents in patients with DKD.
Subject(s)
Cytoskeleton/metabolism , Diabetic Nephropathies/genetics , Fructosediphosphates/metabolism , Phosphofructokinase-1, Type C/genetics , Podocytes/metabolism , RNA, Messenger/metabolism , Adult , Albuminuria , Animals , Cytoskeleton/pathology , Diabetes Mellitus/metabolism , Diabetes Mellitus/pathology , Diabetic Nephropathies/metabolism , Diabetic Nephropathies/pathology , Female , Fructose-Bisphosphate Aldolase/genetics , Fructose-Bisphosphate Aldolase/metabolism , Glycolysis , Hexokinase/genetics , Hexokinase/metabolism , Humans , L-Lactate Dehydrogenase/genetics , L-Lactate Dehydrogenase/metabolism , Male , Mice , Middle Aged , Phosphofructokinase-1, Type C/metabolism , Podocytes/pathology , Pyruvate Kinase/genetics , Pyruvate Kinase/metabolismABSTRACT
The platelet isoform of phosphofructokinase (PFKP) is a ratelimiting enzyme involved in glycolysis that serves an important role in various types of cancer. The aim of the present study was to explore the specific regulatory relationship between PFKP and nonsmall cell lung cancer (NSCLC) progression. PFKP expression in NSCLC tissues and corresponding adjacent tissues was detected using reverse transcriptionquantitative polymerase chain reaction (RTqPCR) and immunohistochemical analysis. PFKP expression in human bronchial epithelial cells (16HBE) and NSCLC cells (H1299, H23 and A549) was also detected using RTqPCR. Cell proliferation was detected by Cell Counting Kit8 and colony formation assays. Transwell invasion and wound healing assays, and flow cytometry were used to detect cell invasion, migration and apoptosis, respectively. The expression levels of glycolysisassociated enzymes (hexokinase2, lactate dehydrogenase A and glucose transporter1), epithelialmesenchymal transitionrelated proteins (Ncadherin, vimentin and Ecadherin) and apoptosisrelated proteins (caspase3 and Bcell lymphoma2) were detected by western blotting. Glucose uptake, lactate production and the adenosine trisphosphate/adenosine diphosphate ratio were measured using the corresponding kits. The results of the present study demonstrated that PFKP expression was upregulated in NSCLC tissues and cells, and PFKP expression was related to lymph node metastasis and histological grade. In addition, overexpression of PFKP inhibited cell apoptosis, and promoted proliferation, migration, invasion and glycolysis of H1299 cells, whereas knockdown of PFKP had the opposite effects. In conclusion, PFKP inhibited cell apoptosis, and promoted proliferation, migration, invasion and glycolysis of NSCLC cells; these findings may lay the foundation for novel treatments of NSCLC.
Subject(s)
Carcinoma, Non-Small-Cell Lung/enzymology , Glycolysis , Lung Neoplasms/enzymology , Neoplasm Proteins/metabolism , Phosphofructokinase-1, Type C/metabolism , A549 Cells , Aerobiosis , Aged , Carcinoma, Non-Small-Cell Lung/pathology , Female , Humans , Lung Neoplasms/pathology , Male , Middle AgedABSTRACT
AIMS: Pathological cardiac remodelling is characterized by cardiomyocyte (CM) hypertrophy and fibroblast activation, which can ultimately lead to maladaptive hypertrophy and heart failure (HF). Genome-wide expression analysis on heart tissue has been instrumental for the identification of molecular mechanisms at play. However, these data were based on signals derived from all cardiac cell types. Here, we aimed for a more detailed view on molecular changes driving maladaptive CM hypertrophy to aid in the development of therapies to reverse pathological remodelling. METHODS AND RESULTS: Utilizing CM-specific reporter mice exposed to pressure overload by transverse aortic banding and CM isolation by flow cytometry, we obtained gene expression profiles of hypertrophic CMs in the more immediate phase after stress, and CMs showing pathological hypertrophy. We identified subsets of genes differentially regulated and specific for either stage. Among the genes specifically up-regulated in the CMs during the maladaptive phase we found known stress markers, such as Nppb and Myh7, but additionally identified a set of genes with unknown roles in pathological hypertrophy, including the platelet isoform of phosphofructokinase (PFKP). Norepinephrine-angiotensin II treatment of cultured human CMs induced the secretion of N-terminal-pro-B-type natriuretic peptide (NT-pro-BNP) and recapitulated the up-regulation of these genes, indicating conservation of the up-regulation in failing CMs. Moreover, several genes induced during pathological hypertrophy were also found to be increased in human HF, with their expression positively correlating to the known stress markers NPPB and MYH7. Mechanistically, suppression of Pfkp in primary CMs attenuated stress-induced gene expression and hypertrophy, indicating that Pfkp is an important novel player in pathological remodelling of CMs. CONCLUSION: Using CM-specific transcriptomic analysis, we identified novel genes induced during pathological hypertrophy that are relevant for human HF, and we show that PFKP is a conserved failure-induced gene that can modulate the CM stress response.
Subject(s)
Cardiomegaly/genetics , Gene Expression Profiling , Myocytes, Cardiac/metabolism , Transcriptome , Ventricular Remodeling/genetics , Animals , Cardiac Myosins/genetics , Cardiac Myosins/metabolism , Cardiomegaly/metabolism , Cardiomegaly/pathology , Cardiomegaly/physiopathology , Cells, Cultured , Disease Models, Animal , Fibrosis , Gene Expression Regulation , Humans , Male , Mice, Inbred C57BL , Mice, Transgenic , Myocytes, Cardiac/pathology , Myosin Heavy Chains/genetics , Myosin Heavy Chains/metabolism , Natriuretic Peptide, Brain/genetics , Natriuretic Peptide, Brain/metabolism , Phosphofructokinase-1, Type C/genetics , Phosphofructokinase-1, Type C/metabolismABSTRACT
Neural tube closure is a critical early step in central nervous system development that requires precise control of metabolism to ensure proper cellular proliferation and differentiation. Dysregulation of glucose metabolism during pregnancy has been associated with neural tube closure defects (NTDs) in humans suggesting that the developing neuroepithelium is particularly sensitive to metabolic changes. However, it remains unclear how metabolic pathways are regulated during neurulation. Here, we used single-cell mRNA-sequencing to analyze expression of genes involved in metabolism of carbon, fats, vitamins, and antioxidants during neurulation in mice and identify a coupling of glycolysis and cellular proliferation to ensure proper neural tube closure. Using loss of miR-302 as a genetic model of cranial NTD, we identify misregulated metabolic pathways and find a significant upregulation of glycolysis genes in embryos with NTD. These findings were validated using mass spectrometry-based metabolite profiling, which identified increased glycolytic and decreased lipid metabolites, consistent with a rewiring of central carbon traffic following loss of miR-302. Predicted miR-302 targets Pfkp, Pfkfb3, and Hk1 are significantly upregulated upon NTD resulting in increased glycolytic flux, a shortened cell cycle, and increased proliferation. Our findings establish a critical role for miR-302 in coordinating the metabolic landscape of neural tube closure.
Subject(s)
Cell Cycle , Glycolysis , MicroRNAs/metabolism , Neural Tube/metabolism , Neurulation , Animals , Cells, Cultured , Hexokinase/genetics , Hexokinase/metabolism , Mice , Mice, Inbred C57BL , MicroRNAs/genetics , Neural Tube/embryology , Phosphofructokinase-1, Type C/genetics , Phosphofructokinase-1, Type C/metabolism , Phosphofructokinase-2/genetics , Phosphofructokinase-2/metabolismABSTRACT
Efficient catabolic metabolism of adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) is essentially required for cancer cell survival, especially in metastatic cancer progression. Epithelial-mesenchymal transition (EMT) plays an important role in metabolic rewiring of cancer cells as well as in phenotypic conversion and therapeutic resistance. Snail (SNAI1), a well-known inducer of cancer EMT, is critical in providing ATP and NADPH via suppression of several gatekeeper genes involving catabolic metabolism, such as phosphofructokinase 1 (PFK1), fructose-1,6-bisphosphatase 1 (FBP1), and acetyl-CoA carboxylase 2 (ACC2). Paradoxically, PFK1 and FBP1 are counter-opposing and rate-limiting reaction enzymes of glycolysis and gluconeogenesis, respectively. In this study, we report a distinct metabolic circuit of catabolic metabolism in breast cancer subtypes. Interestingly, PFKP and FBP1 are inversely correlated in clinical samples, indicating different metabolic subsets of breast cancer. The luminal types of breast cancer consist of the pentose phosphate pathway (PPP) subset by suppression of PFKP while the basal-like subtype (also known as triple negative breast cancer, TNBC) mainly utilizes glycolysis and mitochondrial fatty acid oxidation (FAO) by loss of FBP1 and ACC2. Notably, PPP remains active via upregulation of TIGAR in the FBP1-loss basal-like subset, indicating the importance of PPP in catabolic cancer metabolism. These results indicate different catabolic metabolic circuits and thus therapeutic strategies in breast cancer subsets.